Detection of shed cd31, diagnosis of atherothrombosis and autoimmune disorders, and methods for analyzing signaling pathways

ABSTRACT

The present invention stems from the finding that the extracellular domain of CD31 proteins present on blood leukocytes is shed and released in the circulation as a soluble form of CD31. A method for detecting shed CD31 is further disclosed. The invention therefore relates to a method for detecting a shed ectodomain of a transmembrane protein such as CD31 and to the use of such a method as a diagnostic tool. The invention further provides methods for determining whether a candidate protein is part of a molecular complex.

FIELD OF THE INVENTION

The present invention stems from the finding that the extracellulardomain of CD31 proteins present on blood leukocytes is shed and releasedin the circulation as a soluble form of CD31. A method for detectingshed CD31 is further disclosed. The invention therefore relates to amethod for detecting a shed ectodomain of a transmembrane protein suchas CD31 and to the use of such a method as a diagnostic tool. Theinvention further provides methods for determining whether a candidateprotein is part of a molecular complex.

BACKGROUND

CD31 (PECAM-1)

Immune responses can be controlled by inhibitory immune receptors amongwhich CD31 (PECAM-1), which is expressed exclusively and constitutivelyon cells at the blood-vessel interface.

CD31 consists of a single chain molecule comprising 6 Ig-likeextracellular domains, a short transmembrane segment and a cytoplasmictail containing two ImmunoTyrosine-based Inhibitory Motif (ITIM)s. Thestructure of CD31 is shown in the table below.

Domain Position on SEQ ID No: 1 Signal peptide  1 to 27 Extracellulardomain  28 to 601 First Ig-like extracellular domain  34 to 121 SecondIg-like extracellular domain 145 to 233 Third Ig-like extracellulardomain 236 to 315 Fourth Ig-like extracellular domain 328 to 401 FifthIg-like extracellular domain 424 to 493 Sixth Ig-like extracellulardomain 499 to 591 Juxta-membrane domain 592 to 601 Transmembrane domain602 to 620 Cytoplasmic domain 621 to 738

The consequent putative immunoregulatory properties of CD31 aresupported by the fact that CD31 signaling drives mutual repulsion ofblood leukocytes and modulates the balance between inhibitory andstimulatory signals of both innate and adaptive immune cells. Mechanicalengagement of the distal Ig-like extracellular domains of CD31 inducesoutside-in inhibitory signaling triggered by the phosphorylation of itsITIMs, and the recruitment and activation of SH2-containingphosphatases.

Zehnder et al. (1995, Blood. 85(5):1282-8) identified a CD31 antibodythat inhibited the mixed lymphocyte reaction (MLR) in a specific anddose-dependent manner. They further found that a CD31 peptidecorresponding to the epitope of this antibody, i.e. to the 23membrane-proximal amino acids of CD31, strongly inhibited the MLR. Theyhypothesized that the 23 membrane-proximal amino acids of CD31 is afunctionally important region, and that the CD31 peptide interferes withlymphocyte activation by competing for binding epitopes. However,Zehnder et al. failed to teach whether CD31-mediated signaling isactivated or inhibited by the CD31 peptide.

Chen et al. (1997, Blood. 89(4):1452-9) showed that this peptide delayedonset of graft-versus-host disease (GVHD) and increased long-termsurvival in a murine model of the disease. They hypothesized that theCD31 peptide inhibits a common pathway in T-cell activation. Again, Chenet al. failed to elucidate the role played by the CD31 peptide in T-cellactivation. In particular, these previous works did not assess theputative effect of the peptide on the CD31 signaling cascade and moreprecisely on the phosphorylation state of the CD31 ITIMs.

For a yet unknown mechanism, CD31 is “lost” on certain circulatinglymphocytes. Its loss is observed upon lymphocyte activation and it hasbeen recently shown that the absence of lymphocyte CD31 signaling, inturn, heightens the pathologic immune responses involved in thedevelopment of atherothrombosis.

A soluble form of CD31, due to a variant transcript lacking thetransmembrane segment, has also been reported and therefore it iscurrently thought that the individual amount of circulating CD31 isgenetically determined. Consequently, a number of previous studies haveattempted to find a correlation between plasma levels of soluble CD31and the risk of atherothrombosis or other autoimmune diseases. However,independently of the specific genetic polymorphisms analyzed, datashowed a broad range of plasma CD31 values and the results of thesedifferent studies were contradicting.

There is therefore a need for better understanding the biologicalfunction of CD31. This would allow the provision of better tools for thediagnosis of diseases linked with T-cell activation such as thromboticand autoimmune disorders.

Methods for Analyzing Signaling Pathways

Three different assays are currently available for analyzing signalingpathways: co-immunoprecipitation followed by Western Blot (co-IP/WB),the Cellular Activation of Signaling ELISA assay (CASE) and the CBA Flexset (BD) assay.

In the co-IP/WB assay, cells are lysed and a protein extraction is firstcarried out. The studied protein is then co-immunoprecipitated withproteins with which it is associated and a 2D electrophoresis is carriedout. Finally, the membrane obtained after the Western Blot is hybridizedwith a labelled antibody and the signal is detected. It is thus atime-consuming assay. The membrane may be deshybridized and rehybridizedwith another labelled antibody, but no more than four times. Thus onlyfour parameters may be analyzed with a single sample/membrane. Inaddition, the co-IP/WB assay is a qualitative but not a quantitativeassay. Finally, the skilled in the art must perform two separateco-IP/WB assays if he wishes to compare proteins associated with thestudied protein in a phosphorylated state with those associated with thestudied protein in an unphosphorylated state.

The CASE (Superarray) and Phosphlow (BD) assays are simpler and morerapid than the co-IP/WB assay since the labelled antibody is added to asample comprising permeabilized but not lysed cells. In addition, noblotting is needed. The antibodies are labelled with a fluorophore(Phosphlow) or detected via an enzyme (CASE), the activity of which caneasily be measured. However, these assays do still not allow analyzingmany parameters with a single sample since only a limited number ofenzymes are available. In addition, both assays are much less specificthan the co-IP/WB assay since the studied molecule is not captured andtherefore one cannot determine the interaction between the differentmolecules of the signaling pathway. The Phosphlow and CASE assays onlyallow determining which molecules of a given signaling pathway arepresent in a cell, or present in a phosphorylated state in the cell.

The CBA Flex set (BD) assay allows simultaneous determination ofmultiple signaling molecules but fails to allow the analysis ofmolecular complexes, and therefore, the determination of specificreceptor-dependent signaling cascades is impossible.

There is therefore a need for improved methods for analyzing signalingpathways, which combines the respective advantages of co-IP/WB,Phosphlow/CASE and CBA Flex set.

DESCRIPTION OF THE INVENTION

In this context, it has surprisingly been found that the assumed loss ofCD31 on activated/memory T lymphocytes is actually incomplete andresults from shedding of CD31 between the 5^(th) and the 6^(th)extracellular Ig-like domains. The shed extracellular domain of CD31(further referred to as “shed CD31”) is then released into thecirculation, where it is present together with a soluble splice variantof CD31.

In addition, it has been shown that a high risk of atherothrombosis islinked with the increase of the shed CD31 and decrease of the splicevariant CD31 in the circulation, and not with the total level of thecirculating CD31.

This finding led to the provision of a powerful diagnostic tool. Indeed,since tests that were commercially available so far detected plasma CD31through the use of antibodies directed to CD31 domains 1 to 5, theycould not discriminate between the soluble splice variant of CD31(containing all the 6 extracellular Ig-like domains) and the shed formof CD31 (containing domains 1 to 5 only). On the other hand, thesubtractive method described herein allows discriminating between thetwo forms of soluble CD31 and precisely quantify the proportion of eachof them in a biological sample.

It has been further found that this subtractive method can be adapted inorder to analyze soluble forms of other transmembrane proteins thanCD31, and to analyze molecular complexes and signaling pathways, e.g. todetermine whether a signaling pathway is activated or not, and/or todetermine whether a candidate protein is part of a molecular complex.

The invention therefore provides methods for detecting and/orquantifying a shed ectodomain of a transmembrane protein, methods fordiagnosing whether an individual suffers from a thrombotic or anautoimmune disorder, diagnostic kits, methods for determining whether asignaling pathway is activated, and methods for determining whether acandidate molecule is part of a molecular complex as further describedherein.

The methods according to the invention have many advantages compared toprior art methods such as detection of soluble forms by ELISA. Firstly,they can be infinitely replicated. Indeed, each bead represents anindividual test unit, and the number of acquired beads corresponds tothe number of replicates. ELISA tests are usually carried out induplicate or triplicate. However, with the method according to theinvention, at least 300 replicates (i.e. cytometric beads) can beacquired simultaneously. Secondly, the methods of the invention are nottime-consuming since they can be carried out in about 30 minutes.Thirdly, the quantity of starting material (i.e. of biological sample)that is needed is very low (about 5 μg). Fourthly, several differentsoluble forms can be detected simultaneously within the same biologicalsample. Finally, the methods of the invention are more sensitive thanELISA due to the extended range allowed by fluorescent versuscolorimetric methods.

Detection of Shed Ectodomains

A method allowing differentiating between different soluble isoforms ofa protein has been developed. This method is based on the simultaneoususe of antibodies labelled with cytometric beads and offluorescently-labelled antibodies. The labelled antibodies are thendetected by flow cytometry. Briefly, the sample comprising the solubleisoforms is contacted with a first labelled antibody (referred to as the“capture antibody” or the “signaling antibody”) that binds to allsoluble isoforms to be analyzed. The sample is also contacted withlabelled “discriminating antibodies” that only bind to some of thesoluble isoforms. The sample is then analyzed by flow cytometry and theproportion of each of the isoforms in said sample is calculated.

Therefore, the invention provides a method for detecting a shedectodomain of a transmembrane protein among soluble forms of saidtransmembrane protein in a biological sample, wherein said soluble formsinclude a soluble splice variant of said transmembrane protein andoptionally said shed ectodomain, which comprises the steps of:

-   -   a) providing a first type of bead linked to an antibody which        specifically binds to an epitope located in a region that is        present both on said shed ectodomain and on said splice variant;    -   b) providing at least a second type of bead linked to an        antibody which specifically binds to an epitope located in a        region that is either present on said shed ectodomain and absent        from said splice variant, or present on said splice variant and        absent from said shed ectodomain;    -   c) providing a fluorescently-labelled ligand which specifically        binds to a region that is present both on said shed ectodomain        and on said splice variant;    -   d) contacting said antibodies with a biological sample likely to        contain said soluble forms of said transmembrane protein;    -   e) for each type of bead, measuring the signal obtained with        said florescent label by flow cytometry; and    -   f) comparing the signal obtained for each type of bead.        wherein a difference in the signals measured at step (e)        indicates that the biological sample comprises said shed        ectodomain. In a preferred embodiment, the biological sample is        first contacted with the bead-linked antibodies, the beads are        then recovered and contacted with the fluorescently-labelled        ligand.

In the above method, the shed ectodomain is detected using one singlefluorescently-labelled ligand as a signaling ligand and at least twotypes of bead-linked antibodies as discriminating antibodies.

Alternatively, the shed ectodomains may be detected using one type ofbead-linked ligand as a capture ligand and at least twofluorescently-labelled antibodies as discriminating antibodies.

Such a method for detecting a shed ectodomain of a transmembrane proteinamong soluble forms of said transmembrane protein in a biologicalsample, wherein said soluble forms include a soluble splice variant ofsaid transmembrane protein and optionally said shed ectodomain,comprises the steps of:

-   -   a) providing a bead linked to a ligand which specifically binds        to a region that is present both on said shed ectodomain and on        said splice variant;    -   b) providing a first type of fluorescently-labelled antibody        which specifically binds to an epitope located in a region that        is present both on said shed ectodomain and on said splice        variant;    -   c) providing at least a second type of fluorescently-labelled        antibody which specifically binds to an epitope located in a        region that is either present on said shed ectodomain and absent        from said splice variant, or present on said splice variant and        absent from said shed ectodomain;    -   d) contacting said antibodies with a biological sample likely to        contain said soluble forms of said transmembrane protein;    -   e) for each fluorescently-labelled antibody, measuring the        signal obtained with said florescent label by flow cytometry;        and    -   f) comparing the signal obtained for each fluorescently-labelled        antibody;        wherein a difference in the signals measured at step (e)        indicates that the biological sample comprises said shed        ectodomain. In this method, each type of fluorescently-labelled        antibody is labelled with a different label. Thus the label of        the second type of fluorescently-labelled antibody is different        from the label of the first type of fluorescently-labelled        antibody. In the frame of this method, the bead is preferably        not fluorescently labelled. In a preferred embodiment, the        biological sample is first contacted with the bead-linked        ligand, the beads are then recovered and contacted with the        fluorescently-labelled antibodies.

The above methods for detecting a shed ectodomain of a transmembraneprotein among soluble forms of said transmembrane protein in abiological sample may further comprise the step of calculating theproportion, percentage and/or amount of said soluble forms thatcorresponds to said shed ectodomain, and/or the step of comparing themeasured signals with those obtained with at least one biological samplecomprising known amounts of said shed ectodomain and of said solublesplice variants, and/or the step of calculating the ratio of shedectodomain (or spliced form) to soluble forms (i.e. all soluble isoformsor “total soluble forms”).

As used herein, the term “bead” refers to a cytometric bead for use inflow cytometry. In the method according to the invention, differenttypes of beads refer to beads distinguishable from each other. Suchbeads may for example correspond to BD™ Cytometric Beads commercializedby BD Biosciences (San Jose, Calif.). Beads are well-known in the artand are further described below.

Flow cytometers enable the characterization of particles on the basis oflight scatter and particle fluorescence. In a flow cytometer, particlesare individually analyzed by exposing each particle to an excitationlight, typically one or more lasers, and the light scattering andfluorescence properties of the particles are measured. Particles, suchas molecules, analyte-bound beads, individual cells, or subcomponentsthereof, typically are labelled with one or more spectrally distinctfluorescent dyes, and detection is carried out using a multiplicity ofphotodetectors, one for each distinct dye to be detected. Flowcytometers are commercially available from, for example, BD Biosciences(San Jose, Calif.). Early in the development of flow cytometry, it wasrecognized that various types of ligand binding assays could be carriedout using beads (also referred to as microparticles) coated with onemember of a binding pair. The coated beads and reporters are incubatedwith a sample containing (or suspected of containing) the analyte ofinterest to allow for the formation of bead-analyte-reporter complexes.Analysis by flow cytometry enables both detecting the presence ofbead-analyte-reporter complexes and simultaneously measuring the amountof reporter fluorescence associated with the complex as a quantitativemeasure of the analyte present in the sample. It was also recognizedearly in the development of flow cytometry that the simultaneousanalysis of multiple analytes in a sample could be carried out using aset of distinguishable beads, each type of bead coated with a uniqueanalyte-specific binding agent. The bead set and fluorescently labelledreporter reagents, one for each species of analyte to be detected, areincubated with a sample containing the analytes of interest to allow forthe formation of bead-analyte-reporter complexes for each analytepresent, and the resulting complexes are analyzed by flow cytometry toidentify and, optionally, quantify the analytes present in the sample.Because the identity of the analyte bound to the complex is indicated bythe identity of the bead, multiple analytes can be simultaneouslydetected using the same fluorophore for all reporter reagents.

A number of methods of making and using sets of distinguishablemicroparticles have been described in the literature. These includebeads distinguishable by size, wherein each size microparticle is coatedwith a different target-specific antibody (see e.g. Fulwyler and McHugh,1990, Methods in Cell Biology 33:613-629), beads with two or morefluorescent dyes at varying concentrations, wherein the beads areidentified by the levels of fluorescence dyes (see e.g. European PatentNo. 0 126,450), and beads distinguishably labelled with two differentdyes, wherein the beads are identified by separately measuring thefluorescence intensity of each of the dyes (see e.g. U.S. Pat. Nos.4,499,052 and 4,717,655).

Both one-dimensional and two-dimensional arrays for the simultaneousanalysis of multiple analytes by flow cytometry are availablecommercially. Examples of one-dimensional arrays of singly dyed beadsdistinguishable by the level of fluorescence intensity include the BD™Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) andCyto-Plex™ Flow Cytometry microspheres (Duke Scientific, Palo Alto,Calif.). An example of a two-dimensional array of beads distinguishableby a combination of fluorescence intensity (five levels) and size (twosizes) is the QuantumPlex™ microspheres (Bangs Laboratories, Fisher,Ind.). An example of a two-dimensional array of doubly-dyed beadsdistinguishable by the levels of fluorescence of each of the two dyes isdescribed in Fulton et al. (1997, Clinical Chemistry 43(9):1749-1756).

As used herein, the term “specifically binding” has its usual meaningwithin the art. The fact whether a molecule specifically binds toanother molecule is generally determined by a competitive binding assay.

The biological sample may for example correspond to plasma, blood orurine. The biological sample preferably corresponds to plasma. Mostpreferably, the biological sample is obtained from an individualsuffering from or at risk of suffering from a thrombotic or anautoimmune disorder.

By “ligand” is meant a natural ligand, an antibody or an aptamer. Theligand preferably is an antibody that specifically binds to an epitopelocated in a region that is present both on the shed ectodomain and onthe splice variant.

As used herein, the term “antibody” refers both to monoclonal and topolyclonal antibodies. The antibody is preferably a monoclonal antibody.However, it may also correspond to a polyclonal antibody.

The fluorescently-labelled ligand and/or antibody may be labelled withany fluorescent compound known in the art such as e.g. FITC (FL1), PE(FL2), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7,PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red,violet or uv laser (e.g. Pacific blue, pacific orange). Optimizedmethods include different emission spectra for the beads and thedetecting antibodies. The antibody is preferably directly labelled.However, it may also be indirectly labelled, especially when theantibody is a polyclonal antibody.

As used herein, the term “shed ectodomain of a transmembrane protein”refers to an extracellular portion of a transmembrane protein which hasbeen cleaved by proteolytic processing. Examples of transmembraneproteins for which a shed ectodomain exist include but are not limitedto L-selectin (Asimakopoulos et al., Perfusion, 2000. 15(6): p. 495-9),ICAM-1 (Becker, et al., J Immunol, 1991. 147(12): p. 4398-401), VCAM-1(Garton et al., SJ Biol Chem, 2003. 278(39): p. 37459-64), VCAM-1receptor (Belgore et al., Am J Cardiol, 2001. 87(6): p. 805-7, A9),P-selectin (Dole et al., Thromb Haemost, 2007. 98(4): p. 806-12), CD40(Contin et al. J Biol Chem, 2003. 278(35): p. 32801-9), CD23 (Gu et al.,Blood, 1998. 92(3): p. 946-51), CD21 (Fremeaux-Bacchi et al., IntImmunol, 1998. 10(10): p. 1459-66), HLA-E (Derre et al., J Immunol,2006. 177(5): p. 3100-7), NgR (Ahmed et al., Faseb J, 2006. 20(11): p.1939-41), Hepatocyte growth factor (Wajih et al. Circ Res, 2002. 90(1):p. 46-52), IL-6R (Franchimont et al., Arthritis Rheum, 2005. 52(1): p.84-93), TNFa (Fabris et al., Clin Exp Immunol, 1999. 117(3): p. 556-60),IL-4R (Silvestri et al., Osteoarthritis Cartilage, 2006. 14(7): p.717-9), IL-1R (Beck et al., Mol Immunol, 1994. 31(17): p. 1335-44),transferring receptor (Chitambar et al., Blood, 1991. 78(9): p. 2444-50)and the common gamma chain of immunoglobulins (Meissner et al., Blood,2001. 97(1): p. 183-91).

In a preferred embodiment according to the invention, the transmembraneprotein is CD31.

As used herein, the term “CD31” refers to the platelet/endothelial celladhesion molecule also referred to as CD31 antigen or PECAM-1. Theprotein may be of any origin, preferably of mammalian origin, and mostpreferably of human origin. In human, the gene coding for CD31 islocated at locus 17q23. The sequence of human wild-type CD31 is shown asSEQ ID NO: 1. However, the invention also relates to allelic variantsthereof, and to the homologs thereof in other species.

As shown in FIG. 5 and in the Sequence Listing for SEQ ID NO: 1 (humanCD31), the CD31 protein comprises six extracellular immunoglobulin-like(Ig-like) domains. In addition, CD31 exists not only as a transmembraneprotein but also as a soluble splice variant comprising the sixextracellular Ig-like domains (see Goldberger et al. 1994. J Biol Chem269:17183-17191).

It has unexpectedly been found that soluble forms of CD31 include a shedectodomain comprising the 1^(st), 2^(nd), 3^(rd), 4^(th) and 5^(th)extracelluar Ig-like domains. Thus the 6^(th) extracellular Ig-likedomain is present in the soluble splice variant but absent from the shedectodomain.

Therefore, a preferred embodiment of the invention relates to a methodfor detecting a shed ectodomain of CD31 among soluble forms of CD31 in abiological sample wherein at least two discriminating antibodies areused, one specifically binding to an epitope located in either of thefirst five extracellular immunoglobulin-like domain of CD31, and anotherone specifically binding to an epitope located in the sixthextracellular immunoglobulin-like domain of CD31. The capture orsignaling ligand may for example specifically bind to the first and/orsecond extracellular immunoglobulin-like domains of CD31. the capture ofsignaling ligand is preferably a capture or signaling antibody, whichmay for example specifically bind to an epitope located in the firstand/or second extracellular immunoglobulin-like domains of CD31.

Such antibodies are well-known in the art and may for example correspondto any one of the antibodies listed in the HLDA Antibody Database (seeworld wide web page99.mh-hannover.de/aktuelles/projekte/hlda7/hldabase/CD31.htm).

As used herein, the term “epitope located in the sixth extracellularimmunoglobulin-like domain of CD31” refers to an epitope located withinamino acids 499 to 601 of SEQ ID NO: 1, i.e. within the sixthextracellular immunoglobulin-like domain of CD31 (amino acids 499 to 591of SEQ ID NO: 1) and/or within the juxta-membrane region (amino acids592 to 601 of SEQ ID NO: 1). Preferably, said epitopes are locatedwithin amino acids 499 to 591, 524 to 601 or 524 to 538 of SEQ ID NO: 1.In a preferred embodiment, the antibody which specifically binds to anepitope located in the sixth extracellular immunoglobulin-like domain ofCD31 corresponds to the PECAM 1.2 antibody (Invitrogen, San Diego,Calif.), the PECAM1.1 antibody, or the HC1/6 antibody (Serotec,Kidlington, UK). The PECAM 1.2 antibody has been described e.g. in Yanet al. (Cell Adhes Commun 3:45-66).

As used herein, the term “epitope located in either of the first fiveextracellular immunoglobulin-like domain of CD31” refers to an epitopelocated within amino acids 28 to 493 of SEQ ID NO: 1, most preferablywithin amino acids 34 to 493 of SEQ ID NO: 1. In preferred embodimentsaccording to the invention, said epitope is located in the fifthextracellular immunoglobulin-like domain of CD31, or in the first and/orsecond extracellular immunoglobulin-like domain of CD31.

As used herein, the term “epitope located in the fifth extracellularimmunoglobulin-like domain of CD31” refers to an epitope located withinamino acids 424 to 493 of SEQ ID NO: 1. Preferably, said epitopes arelocated within amino acids 448 to 470 of SEQ ID NO: 1. In a preferredembodiment, the antibody which specifically binds to an epitope locatedin the fifth extracellular immunoglobulin-like domain of CD31corresponds to the MEM-05 antibody (Zymed Laboratories, South SanFrancisco, Calif.).

As used herein, the term “epitope located in the first and/or secondextracellular immunoglobulin-like domain of CD31” refers to an epitopelocated within amino acids 34 to 233 of SEQ ID NO: 1. An epitope locatedin the first extracellular immunoglobulin-like domain of CD31 ispreferably located within amino acids 49 to 68 of SEQ ID NO: 1. Anepitope located in the first extracellular immunoglobulin-like domain ofCD31 is preferably located within amino acids 166 to 187 of SEQ IDNO: 1. In a preferred embodiment, the antibody which specifically bindsto an epitope located in the first two extracellular immunoglobulin-likedomains of CD31 corresponds to the WM59 antibody (domain 2, BD, SanJose, Calif.), to the JC70A antibody (domain 1, DAKO, Glostrup,Denmark), or to the 9G11 antibody (domain 1, R&D systems, Minneapolis,USA). The WM59 and JC70A antibodies have been described e.g. in Fawcettet al. (J Cell Biol 128:1229-1241).

Said soluble forms of the transmembrane protein may include additionalsoluble forms corresponding to e.g. additional soluble splice variants,additional shed ectodomains, or to variants generated by proteolyticprocessing. When the soluble forms include at least three soluble forms,more than two discriminating antibodies may be used. Within thisembodiment, the discriminating antibodies are chosen in such a way as todiscriminate between the soluble forms, and the capture or signalingantibody specifically binds to an epitope located in a region that ispresent on all said soluble forms.

Data from five plasma samples showed that most circulating shed CD31comprises Ig-like domains 1 to 5. However, Ig-like domain 5 could beabsent in up to 16% of plasma CD31, indicating that more that onecleavage site may exist in the extracellular CD31 domains. Therefore, amore accurate test for differentiating between all soluble forms of CD31would include also detection of Ig-like domain 1, in addition todetection of Ig-like domains 5 and 6 of CD31. Thus the method fordetecting a shed ectodomain of CD31 among soluble forms of CD31 in abiological sample may comprise the use of three discriminatingantibodies, which specifically binds to epitopes located in the firstand/or second extracellular immunoglobulin-like domain of CD31, in thefifth extracellular immunoglobulin-like domain of CD31, and in the sixthextracellular immunoglobulin-like domain of CD31, respectively. In thisembodiment, the capture or signaling antibody must specifically bind toan epitope located in the first and/or second extracellularimmunoglobulin-like domains of CD31.

The method for detecting of a shed ectodomain of CD31 may furthercomprise the step of calculating the proportion, percentage and/oramount of said soluble forms that corresponds to said shed ectodomain ofCD31, and/or the step of comparing the measured signals with thoseobtained with at least one biological sample comprising known amounts ofsaid shed ectodomain and of said soluble splice variants. Thecalculation can for example be performed as described e.g. in theparagraph entitled “subtractive measurement of soluble CD31” inExample 1. The method for detecting of a shed ectodomain of CD31preferably comprises the step of calculating the ratio of shedectodomain to total soluble forms, i.e. the ratio of shed CD31 to allsoluble isoforms (soluble isoform comprising domains 1-6, solubleisoform comprising domains 1-5, and soluble isoform comprising domains1-2).

FIG. 4 illustrates a method in accordance with the invention for thedetection and quantification of the shed ectodomain of CD31. Three typesof beads, linked to antibodies specifically binding to epitopes locatedin the 1^(st), the 5^(th) or the 6^(th) extracellular Ig-like domainrespectively, are used. The fluorescently labelled antibody specificallybinds to an epitope located on the 1^(st) and 2^(nd) extracellularIg-like domains. A simple subtractive calculation based on measurementof the intensity of the signal obtained for each bead allows quantifyingthe respective proportion of each of the soluble forms of CD31:

-   -   Soluble isoform comprising domains 1-6=aCD31_(d6)    -   Soluble isoform comprising domains 1-5=aCD31_(d5)−aCD31_(d6)    -   Soluble isoform comprising domains        1-2=aCD31_(d1)−(aCD31_(d5)+aCD31_(d6))

The above methods aim at detecting shed ectodomains which are soluble ina biological fluid. However, these methods may be adapted to detect ashed isoform of a transmembrane protein (e.g. CD31^(shed)) on a cellexpressing said transmembrane protein. In that case, the discriminatingantibodies specifically bind to epitopes that are located either in aregion that is present both on the shed isoform and on the full-lengthtransmembrane protein (e.g. Ig-like domain 6), or in a region that ispresent either on the shed isoform or on the full-length transmembraneprotein (e.g. Ig-like domains 1-2). This aspect of the invention isillustrated for CD31 by Example 2, FIG. 1 and FIG. 2 a.

In another preferred embodiment according to the invention, thetransmembrane protein is GPVI.

As used herein, the term “GPVI” refers to the platelet glycoprotein VI.The protein may be of any origin, preferably of mammalian origin, andmost preferably of human origin. The sequence of human wild-type GPVI isshown in SwissProt Acession No. Q9HCN6. The term “GPVI” encompasses thewild-type sequence, variants thereof such as splice and allelicvariants, and homologs thereof in other species.

The spliced form of GPVI comprises the cytoplasmic tail and cantherefore be detected using specific antibodies such as those raisedagainst a maltose-binding protein (MBP)-GPVI cytoplasmic tail fusionprotein (see e.g. Suzuki-Inoue et al. J Biol. Chem. 2002 277:21561-66).On the contrary, the shed form of GPVI comprises all the ectodomains.Example of suitable anti-ectodomain antibodies include clones 11A12,6B12, 3j24.2 and 9O12.2 disclosed in WO/2001/000810). The GPVI naturalligand convulxin binds both to the spliced form and to the shed form ofGPVI.

Therefore, the detection of shed ectodomains of GPVI can be carried outusing can be performed using either:

-   -   anti-tail and anti-ectodomain antibodies linked to different        types of beads as discriminating antibodies, and fluorescently        labeled convulxin as a signaling ligand, or    -   a convulxin-coupled bead as a capture ligand, differently        labeled fluorescent anti-tail and anti-ectodomain antibodies as        discriminating antibodies.

It is also understood that the above methods may be used for detecting asoluble isoform of a protein even if said soluble form is not a shedectodomain of a transmembrane protein. The methods according to theinventions can be used for detecting a soluble form of any proteinexisting as at least two different soluble forms, e.g. existing as twosoluble splice variants. In other terms, they allow detecting anddistinguishing between different soluble forms of a protein.

The methods according to the invention are illustrated by the specificexample of CD31. However, the skilled in the art can easily adapt thismethod to another protein of his own choice. Depending on the structureof the soluble domains, the skilled in the art will choose antibodiesrecognizing regions present on or absent from only one of the solubleforms. In addition, the skilled in the art will choose antibodies insuch a way as to allow simultaneous binding of several antibodies. Suchantibodies can easily be chosen by ascertaining that the antibodies donot recognize overlapping epitopes. Therefore, a preferred embodiment ofthe invention is directed to a method in which the capture or signalingantibody and the different discriminating antibodies do notcross-compete with each other. Methods for determining whetherantibodies cross-compete with each other are well-known in the art andinclude e.g. the method described by Blanchard et al. (1997,International immunology, 9(12):1775-1784).

In a specific embodiment, methods comprising the step of fragmenting(e.g. by enzymatic digestion) the proteins that are present in thebiological sample are excluded from the scope of the present invention.

The above methods for detecting shed ectodomains and/or shed isoforms ofa transmembrane protein find use both in analytical and diagnosticapplications. Some of the diagnostic applications are described in moredetails in the paragraph herebelow.

Diagnostic Methods and Drug Monitoring

It has been found that a high risk of atherothrombosis is linked withthe presence of shed CD31 in the circulation, and not with the amount oftotal circulating soluble CD31.

The invention therefore provides a method for diagnosing whether anindividual suffers, or is at risk of suffering, from a thrombotic or anautoimmune disorder, which comprises the step of detecting a shedectodomain of CD31 in a biological sample of said individual, whereinthe presence of said shed ectodomain of CD31 indicates that saidindividual suffers from or is at risk of suffering from said thromboticor autoimmune disorder. Where shed ectodomain of CD31 is detectable, themethod may further comprise the step of calculating the ratio of shedectodomain to total soluble forms. Indeed, it has been found that thisratio has a very good predictive value (see Example 7).

Based on this diagnosis, an appropriate treatment regimen may bedesigned for said individual. Preferably, said shed ectodomain of CD31comprises the 1^(st), 2^(nd), 3^(rd), 4^(th) and 5^(th) extracellularIg-like domains but lacks the 6^(th) extracellular Ig-like domain.

As used throughout the present specification, the term “thromboticdisorder” includes but is not limited to atherothrombosis,atherosclerosis, acute coronary syndrome, ischemic stroke, peripheralarterial disease and abdominal aortic aneurysm. Preferably, thethrombotic disorder is atherothrombosis.

As used throughout the present specification, the term “autoimmunedisorder” includes but is not limited to rheumatoid arthritis (RA),spondyloarthritis, multiple sclerosis (MS), inflammatory bowel disease(IBD), systemic lupus erythematodes (SLE), Graves' disease and diabetesmellitus.

The detection of said shed ectodomain of CD31 may be performed accordingto any method known in the art. It may for example be detected by one ofthe methods described in the above paragraph. Alternatively, thedetection may be performed by an ELISA assay. It is however preferred todetect the shed CD31 ectodomain according to a method according to theinvention, since such methods are easier to set up, more rapid and moresensitive.

The biological sample may for example correspond to plasma, blood orurine. The biological sample preferably corresponds to plasma.

The diagnostic method in accordance with the invention may be repeatedat least at two different points in time in order to monitor theprogression of a thrombotic or an autoimmune disorder in the individualand/or to assess the severity of said disorder in said individual,and/or to monitor the response of the individual to a drug.

As used herein, events occurring at “a different (or later) point intime” refer to events occurring at an interval of at least 1 hour.Preferably, the events occur at an interval of at least 6 hours, 12hours, 1 day, one week, two weeks or one month.

The invention also pertains to the use of an antibody which specificallybinds to an epitope located in the sixth extracellularimmunoglobulin-like domain of CD31 for diagnosing whether an individualsuffers, or is at risk of suffering, from a thrombotic or autoimmunedisorder.

The invention further provides a method for diagnosing whether anindividual suffers, or is at risk of suffering, from atherothrombosis,which comprises the steps of:

-   -   a) providing a biological sample of said individual;    -   b) detecting the shed ectodomain of CD31, for example according        to any of the methods described in the above paragraph entitled        “detection of shed ectodomains”, in said biological sample; and    -   c) correlating the result of step (a) with a risk of suffering        from atherothrombosis;        wherein the presence of said shed ectodomain of CD31 in said        biological sample indicates that said individual suffers from or        is at risk of suffering from atherothrombosis. Preferably, said        shed ectodomain of CD31 comprises the 1^(st), 2^(nd), 3^(rd),        4^(th) and 5^(th) extracellular Ig-like domains but lacks the        6^(th) extracellular Ig-like domain.

The presence of at least 50%, 60%, 65%, 70%, 75%, 80%, 90% or 95% ofshed ectodomain in the soluble forms of CD31 indicates that saidindividual suffers from or is at risk of suffering from a thrombotic oran autoimmune disorder such as e.g. atherothrombosis.

In a preferred embodiment, the amount and/or percentage of shed CD31 ina biological sample of said individual to be diagnosed is compared tothe amount and/or percentage of shed CD31 in a biological sample of ahealthy individual.

High levels of CD31 soluble splice variants associated with low levelsof shed CD31 indicates that the individual to be diagnosed suffers fromnon specific chest pain, eventually associated with carotid plaques. Aslight increase of shed CD31 levels associated with normal or reducedlevels of CD31 soluble splice variants indicates that the individual tobe diagnosed suffers from atherosclerosis. An important increase of shedCD31 levels associated with undetectable amounts of CD31 soluble splicevariants indicates that the individual to be diagnosed suffers fromatherothrombosis.

The diagnostic methods in accordance with the invention may be used e.g.to determine whether an individual suffers from a thrombotic or anautoimmune disorder, to assess the severity of a thrombotic or anautoimmune disorder in an individual, to pronostic the risk of majorcardiovascular events, such as recurrence of a myocardial infarction, todesign a treatment regimen, to monitor the progression of a thromboticor an autoimmune disorder in a patient, to predict and to monitor theresponse of a patient to a drug and/or to adjust the treatment of apatient.

When the diagnostic method in accordance with the invention is used tomonitor the progression of a disorder, to assess the severity of adisorder, to monitor the response to a drug and/or to adjust thetreatment of a patient, it carried out on biological samples taken froma given patient at different points in time. Biological samples may forexample be taken each month in order to follow the patient's response toa treatment. Based on these analyses, the treatment may then beadjusted. It may for example be decided to change the drug, or to adjustthe dosage of the drug in order to enhance its efficacy and/or minimizethe side effects. Such a drug monitoring is especially advisable inlong-term treatments, for example when a immunosuppressant compound isadministered to a patient. Detecting shed CD31 can be used to monitorthe inflammatory response in the patient, and thus to determine theminimal effective dose of drug that can be administered to the patient.

The invention therefore provides a method for monitoring the progressionof a thrombotic or an autoimmune disorder, and/or for assessing theseverity of a thrombotic or an autoimmune disorder in an individual,and/or for monitoring the response of a patient to a drug comprising thesteps of:

-   -   a) providing a first biological sample of said patient;    -   b) detecting shed ectodomains of CD31 in said first biological        sample;    -   c) providing at least one second biological sample of said        patient, wherein said at least one second biological sample has        been taken from said patient at a later point in time than the        first biological sample;    -   d) detecting shed ectodomains of CD31 in said at least one        second biological sample;    -   e) comparing the results obtained at steps (b) and (d).

Several different biological samples, taken from the same patient atdifferent points in times, may be used at steps (c), (d) and (e). Forexample, in the frame of a long-term treatment of the patient,biological samples may be taken from the patient at regular intervals(e.g. each month, every two months or twice a year).

Such a method for monitoring the progression of a thrombotic or anautoimmune disorder; and/or to assess the severity of said disorder;and/or for monitoring the response of a patient to a drug may furthercomprise a step (f) of designing a treatment regimen for said patientbased on the results of step (e).

In the frame of drug monitoring, the biological sample of step (a) ispreferably taken before onset of the treatment of the patient, and thebiological sample of step (c) after onset of the treatment. A decreaseof shed CD31 levels measured at step (d) as compared to shed CD31 levelsmeasured at step (b) indicates that the drug is effective for treatingsaid patient.

More specifically, the invention relates to a method for monitoring theresponse of a patient suffering from a thrombotic or autoimmune disorderto a drug, said method comprising the steps of:

-   -   a) detecting shed ectodomains of CD31 in a biological sample of        said patient before and after onset of a treatment of said        patient with said drug;    -   b) comparing the levels of shed ectodomains of CD31 detected at        step (a); and, optionally,    -   c) correlating a difference in said levels of shed ectodomains        of CD31 with the effectiveness of the drug for treating said        patient.

A decrease in the levels of shed ectodomains of CD31 after onset of thetreatment compared with the levels of shed ectodomains of CD31 beforeonset of the treatment indicates that the patient responds to said drug,and that said drug is effective for treating said patient. Preferably,the decrease is of at least 5, 10, 25, 50, 75 or 90%. Conversely, if nosignificant difference in the levels of shed ectodomains of CD31 isfound at step (b), or if an increase in the levels of shed ectodomainsof CD31 after onset of the treatment is found at step (b), the patientdoes not respond to said drug and the drug is not effective for treatingsaid patient.

The invention further pertains to the use of an antibody whichspecifically binds to an epitope located in the sixth extracellularimmunoglobulin-like domain of CD31 for monitoring the progression of athrombotic or an autoimmune disorder in a patient, and/or for monitoringthe response of a patient to a drug.

Diagnostic Kits

The invention also contemplates diagnostic kit comprising:

-   -   a) a fluorescently-labelled antibody that specifically binds to        an epitope located in a region that is present both on said shed        ectodomain and on soluble said splice variant.    -   b) a first type of bead linked to an antibody that specifically        binds to an epitope located in a region that is either present        on said shed ectodomain and absent from said soluble splice        variant, or present on said soluble splice variant and absent        from said shed ectodomain; and    -   c) a second type of bead linked to an antibody that specifically        binds to an epitope located in a region that is present both on        a shed ectodomain of a transmembrane protein and on a soluble        splice variant of said transmembrane protein.

Such kits can be used, e.g., in the diagnostic methods according to theinvention, and/or in drug choice, and/or in drug monitoring.

In a preferred embodiment, the transmembrane protein is CD31. Thefluorescently-labelled antibody and the antibody linked to the firsttype of bead preferably specifically bind to an epitope located ineither of the first five extracellular immunoglobulin-like domain ofCD31. The antibody linked to the second type of bead preferablyspecifically binds to an epitope located in the 6^(th) Ig-like domain.

Such a diagnostic kit may for example comprise:

-   -   a fluorescently-labelled antibody specifically binding to an        epitope located in the first and/or second extracellular        immunoglobulin-like domains of CD31 (e.g. a labelled WM59        antibody, a labeled 9G11 antibody, or a labelled JC70A antibody,        DAKO,);    -   a first type of bead linked to an antibody which specifically        binds to an epitope located in the sixth extracellular        immunoglobulin-like domain of CD31 (e.g. the PECAM 1.2        antibody);    -   one or both of:        -   i) a second type of bead linked to an antibody which            specifically binds to an epitope located in the fifth            extracellular immunoglobulin-like domain of CD31 (e.g. the            MEM-05, PECAM 1.1 or HC1/6 antibody); and        -   ii) a third type of bead linked to an antibody which            specifically binds to an epitope located in the first and/or            second extracellular immunoglobulin-like domain of CD31            (e.g. JC70A, 9G11 or WM59 antibody).

In a specific embodiment, the kit comprises a first type of bead linkedto the PECAM 1.2. antibody, a second type of bead linked to the MEM-05antibody, a fluorescently labelled WM-59 antibody (e.g. PE-WM-59), andoptionally a third type of bead linked to the JC70A antibody.

Other diagnostic kits according to the invention comprise:

-   -   a) a bead linked to an antibody that specifically binds to an        epitope located in a region that is present both on a shed        ectodomain of a transmembrane protein and on a soluble splice        variant of said transmembrane protein;    -   b) a first type of fluorescently-labelled antibody that        specifically binds to an epitope located in a region that is        either present on said shed ectodomain and absent from said        soluble splice variant, or present on said soluble splice        variant and absent from said shed ectodomain; and    -   c) a second type of fluorescently-labelled antibody that        specifically binds to an epitope located in a region that is        present both on said shed ectodomain and on soluble said splice        variant, wherein the label of said second fluorescently-labelled        antibody is different from the label of said first        fluorescently-labelled antibody; and, optionally    -   d) a third type of fluorescently-labelled antibody that        specifically binds to an epitope located in a region that is        present both on said shed ectodomain and on soluble said splice        variant, wherein the label of said third fluorescently-labelled        antibody is different from the label of said first and second        fluorescently-labelled antibodies.

In a preferred embodiment, the transmembrane protein is CD31. The secondfluorescently-labelled antibody and the antibody linked to the beadpreferably specifically bind to an epitope located in either of thefirst five extracellular immunoglobulin-like domain of CD31. The firstfluorescently-labelled antibody preferably specifically binds to anepitope located in the 6^(th) Ig-like domain.

Another preferred embodiment is directed to a kit comprising:

-   -   a bead linked to an antibody specifically binding to an epitope        located in the 1^(st) extracellular Ig-like domain (e.g. a        Pecam1.3, 9G11 or a JC70A antibody);    -   a first type of fluorescently-labelled antibody specifically        binding to an epitope located in the 6th extracellular Ig-like        domain (e.g. a labelled PECAM 1.2 antibody such as FITC-PECAM        1.2);    -   one or both of:        -   a second type of fluorescently-labelled antibody            specifically binding to an epitope located in the 1^(st) or            2^(nd) extracellular Ig-like domain (e.g. a labelled WM59 or            L133 antibody such as PE-WM59 or PE-L133); and        -   a third fluorescently labeled antibody which specifically            binds to an epitope located in the 5^(th) extracellular            Ig-like domain (e.g. the MEM-05 or PECAM 1.1 or HC1/6            antibody);

Fluorescently labeled antibody may include fluorophores exited by ablue-laser and emitting in the FITC, PE, PerCP or PE-tandem channels, orexcited by a red laser and emitting in the Cy5/APC or APC-tandemchannels, or by a violet laser and emitting in the pacific blue orpacific orange channels or by a UV laser and emitting in the Quantum Dotchannels);

These kits may additionally comprise other components such as e.g.reagents and/or instructions.

The kits may also comprise samples comprising a known amount of shedectodomain of CD31 and/or of soluble splice variants, samples fromhealthy individuals and/or samples from individuals suffering fromatherosclerosis or atherothrombosis.

Methods for Analyzing Signaling Pathways

The above methods of detecting shed ectodomains may be adapted for otherpurposes such as the analysis of protein/protein interaction and ofsignaling pathway. This method is easier, more rapid and more powerfulthan co-IP/WB, more specific than CASE/Phosphlow and CBA Flex set, andactually combines the advantages of these techniques.

The invention therefore provides a method of determining whether acandidate molecule is member of a molecular complex which comprises thesteps of:

-   -   a) providing a bead linked to an antibody that specifically        binds to a member of said molecular complex;    -   b) contacting said bead with a biological sample containing said        molecular complex;    -   c) contacting said beads contacted with the biological sample        with at least one type of fluorescently-labelled antibody that        specifically binds to said candidate molecule; and    -   d) detecting the fluorescence by flow cytometry;

wherein:

-   -   the detection of a signal at step (d) indicates that said        candidate molecule is a member of said molecular complex; and    -   if more than one type of fluorescently-labelled antibody is used        at step (c), said antibodies are labelled with different        fluorescent labels.

The method according to the invention allows performing severaldeterminations simultaneously, using the same biological sample.Therefore, the above method may be carried out for at least 2, 3, 4, 5,6, 7 or 10 candidate molecules simultaneously. In such a case, step (c)comprises contacting said beads contacted with the biological samplewith fluorescently-labelled antibodies that specifically bind to each ofsaid at least 2, 3, 4, 5, 6, 7 or 10 candidate molecules. Each of thesefluorescently-labelled antibodies is labelled with a fluorescent labelthat is different from the other fluorescent labels.

The biological sample preferably corresponds to soluble or solubilizedmolecules, preferably derived from a cell. The biological sample may forexample correspond to a cell lysate. A protocol for preparing such acell lysate is for example described in Example 5. Cells may for examplebe lysed on ice for 30 minutes with a RIPA buffer containing a cocktailof protease and phosphatase inhibitors. Alternatively, soluble orsolubilized molecules may for example be prepared by freezing, thawingand crushing cells.

The above method allows gathering multiple information relating to agiven molecular complex and/or a given signaling pathway. As usedherein, a “molecular complex” refers to a complex of associatedmolecules. The molecules may for example correspond to proteins, lipids,sugars or nucleotides. In a preferred embodiment, the molecular complexis a protein complex and the members of said protein complex areproteins.

The method according to the invention allows for example determining theproteins that are associated with a given protein under specificconditions, or determining whether a given protein is phosphorylated ornot (by using a fluorescently-labelled antibody specifically recognizingthe phosphorylated isoform of the protein). The candidate molecule maythus correspond e.g. to a protein that is associated to thebead-captured member of the molecular complex only under certainconditions, a protein that is phosphorylated only under certainconditions, or to a protein for which it is not known whether it is amember of said molecular complex. The method according to the inventionalso allows analyzing a complex of glycoproteins and of protoglycans ofthe extracellular matrix, or a complex of sugars, lipids and proteinsinvolved in thrombus formation.

The antibodies used at step (c) may specifically bind to all isoforms ofa protein likely to be member of said molecular complex, or only to ashed, spliced or phosphorylated isoform of said protein.

Alternatively, the antibodies used at step (c) may bind tophosphorylated amino acids such as phosphotyrosin, phosphoserin orphosphothreonin, irrespective of the primary sequence of the protein.

The antibodies used at step (c) may also specifically bind tonucleotides (DNA or RNA sequences, cyclic nucleotides such as AMPc orGMPc), sugars (e.g. sialic acid), lipids involved in signaling pathways(e.g. steroid hormones, inositol, phosphatidylinositol, leukotrienes,prostaglandins, thomboxane, gangliosides, caveolin, ceramides, etc),lipopolysaccharides involved in TLR-mediated signaling, and glycolipids(e.g. galactocerebroside, which is involved in oligodendrocytesignaling).

The molecular complex may include e.g. membrane receptors, extracellularligands and intracellular proteins such as transduction proteins. Mostpreferably, the candidate molecule is an intracellular molecule, e.g. anintracellular protein.

Examples of molecular complexes that can be analyzed using this methodinclude those given in the table below. The protein captured with thebead-linked antibody may be any one of the members of the molecularcomplex.

Associated Associated Membrane intracellular intracellular Signalingassociated proteins proteins pathway proteins Phosphoproteins(activator) (inhibitor) CD3 signaling CD31, CD3, P56LcK, Zap70, NFkB,CsK, (T cell) CD45, CD28, phosphoITAM of ERK, IkBK, PTPase CTLA4, SLAM,the TCR (CD3ζ) RhoA . . . CD31 IgM signaling IgM, CD19, Iga/b ITAM, RAS,IKK, SHP1, SHIP, (B cell) CD22 SYK, GRB2, PKCb, RAC PTEN, BTK, PI3Kcleaved Notch IL-6R IL-6R, Gp130 IL-6 receptor (4 Jak1, Stat3, SHP2,(cytokine tyrosins) Gab, ERK, SOCS3 signaling) MAPK b-integrinBe-integrin, FAK a-actinin, Sos, signaling Talin, Paxillin, GRb2, Ras,PTPase (cancer Fyn, FAK Raf1,MEK1/2, research) ERK1/2 . . . C83signaling C83 (after GSK3 Cdk5/p25, (after shedding), b- Cdk5/p35shedding) secretase, a- (Alzheimer secretase, C99 research) (left on byb- secretase) APP (on whole molecule) FasL FasL, FADD, p53, Bax, BCL-signaling TRADD, TNF- 2, Apaf-1 , (Apoptosis) R1, RIP, MDM2, c-MycReaper, Caspases, FLIP

In a preferred embodiment, the member of said molecular complex capturedwith the bead-linked antibody is CD31.

The fluorescently-labelled antibody may for example correspond to anantibody that specifically binds to a CD31 protein comprising aphosphorylated tyrosine at position 686. Alternatively, thefluorescently-labelled antibody may for example correspond to anantibody that specifically binds to a phosphorylated isoform of anintracellular protein which is part of the CD31 signaling pathway, or toa transduction protein which is part of the CD31 signaling pathway. Suchproteins are described e.g. in Newman and Newman (2003 ArteriosclerThromb Vasc Biol 23:953-964) and/or in Newton-Nash and Newman (1999. JImmunol 163:682-688). Such antibodies may for example bind to a CD31protein comprising the sixth extracellular immunoglobulin-like domain(e.g. PECAM 1.2), a CD31 protein comprising the second extracellularimmunoglobulin-like domain (e.g. WM59), CD3, CD28, SHP2, CD45, CTLA4,SLAM, p56LcK, CD3ζ, Zap70, NFkB, ERK, IkBK, RhoA, CsK and PTPase.

The invention further provides a kit for analyzing a molecular complexwhich comprises:

-   -   i) a bead linked to an antibody that specifically binds to a        member of said molecular complex; and    -   ii) at least one type of fluorescently-labelled antibody that        specifically binds to another member of said molecular complex.        wherein, if the kit comprises more than one type of        fluorescently-labelled antibody, said antibodies are labelled        with different fluorescent labels.

More specifically, the invention further provides a kit comprising:

-   -   a bead-linked antibody that specifically binds to an epitope        located in the sixth extracellular immunoglobulin-like domain of        CD31 (e.g. PECAM 1.2); and    -   at least one fluorescently-labelled antibody that specifically        binds to a protein selected from the group consisting of a CD31        protein comprising a phosphorylated tyrosine at position 686        (e.g. a labelled rabbit anti-CD31 phospho-tyrosine 686        polyclonal antibody, which may be labelled e.g. with        AlexiaFluor®488-conjugated (Fab′)₂ fragments); a CD31 protein        comprising the sixth extracellular immunoglobulin-like domain        (e.g. PECAM 1.2), a CD31 protein comprising the second        extracellular immunoglobulin-like domain (e.g. WM59), CD3, CD28,        SHP2, CD45, CTLA4, SLAM, p56LcK, CD3ζ, Zap70, NFkB, ERK, IkBK,        RhoA, CsK and PTPase.

The invention also provides kits for analyzing other molecularcomplexes. Such kits may for example comprise a bead-linked antibody andat least one fluorescently-labelled antibody, wherein each antibodyspecifically binds to a protein selected from the group consisting of:

-   -   IgM, CD19, CD22, Iga/b ITAM, SYK, GRB2, BTK, PI3K, RAS, IKK,        PKCb, RAC, SHP1, SHIP, PTEN and cleaved Notch;    -   IL-6R, Gp130, Jak1, Stat3, Gab, ERK, MAPK, SHP2, SOCS3;    -   Beta-integrin, Talin, Paxillin, Fyn, FAK, a-actinin, GRb2, Ras,        Raf1, MEK1/2, ERK1/2, Sos and PTPase;    -   C83 (after shedding), b-secretase, a-secretase, C99 (left on by        b-secretase) APP (on whole molecule), GSK3, Cdk5/p25, Cdk5/p35;        or    -   FasL, FADD, TRADD, TNF-R1, RIP, Reaper, Caspases, FLIP, p53,        Bax, BCL-2, Apaf-1, MDM2, c-Myc.

The kits according to the invention may further comprise afluorescently-labelled antibody that specifically binds tophosphoTyrosins or to phosphoSerin/Threonins.

The different antibodies used in the methods and kits for analyzing amolecular complex do preferably not cross-compete with each other.

All references cited herein, including journal articles or abstracts,published or unpublished patent application, issued patents or any otherreferences, are entirely incorporated by reference herein, including alldata, tables, figures and text presented in the cited references.

Although having distinct meanings, the terms “comprising”, “having”,“containing’ and “consisting of” have been used interchangeablythroughout this specification and may be replaced with one another.

The invention will be further evaluated in view of the followingexamples and figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a representative example of 10-color flow-cytometryanalysis of human peripheral blood cells from a healthy donor. Isotypecontrols of antibodies anti-CD31 dom1 and anti-CD31 dom6 are shown inthe insets. Lymphocytes, Monocytes and Granulocytes were gated withinthe FSC/SSC scatter. B (CD20 A700+) and T (CD3 PE-TR+) lymphocytes wereidentified and gated within the “Lymphocytes” and CD8+ (PerCP) and CD4+(APC) subpopulations were gated within T lymphocytes. CD8+ and CD4+ Tcells were further analyzed for the expression of HLA-DR and CD45RA andaccordingly subdivided in activated (1), memory (2) and naïve (3) cells.All leukocytes were positive for CD31 dom6. Lack of dom1 increased fromnaïve (3) to memory (2) to activated (1) T cells.

FIG. 2 shows that the apparent loss of CD31 on lymphocytes is due to itsextracellular shedding. a. Solubilized cell membrane-bound CD31molecules were extracted from cultured Jurkat CD4+ T cells and coupledto fluorescent beads. The percentage of dom1-bead-bound molecules is <6%in resting conditions and >99% 5′ after TCR engagement. b. Most solubleCD31 in culture supernatant (□) of TCR-activated T cells and in humanplasma (

) consists of a single truncated fragment comprising dom1-dom5 andlacking dom6. Negligible levels of truncated CD31 lacking both dom5 anddom6 could be detected only in plasma.

FIG. 3 shows that a peptide homotypic of the residual extracellularfragment on CD31^(shed)T induces CD31-ITIM phosphorylation. a.Proliferative response to TCR engagement of human peripheral bloodmononuclear cells in the presence of increasing doses of CD31 peptide551-574. *p<0.05 vs dose “0”. b. Flow cytometry assessment of 686ITIMphosphorylation on solubilized membrane-bound CD31 from cultured JurkatCD4+ T cells. Solubilized proteins were captured by E9-PECAM-1.2 (dom6)functional CBA beads and detection was carried out by anti-pY686 rabbitsera followed by AlexaFluor®488-anti-rabbit secondary antibody. Thehistogram shows the Median Fluorescent Intensity (MFI)±the % of thevariability coefficient (CV %) of Alexafluor®488 (pY686) over 2000E9-PECAM-1.2 acquired beads. Pervan=positive control (pervanadate);CD3/CD28=anti-CD3 and anti-CD28 antibodies (1 μg/ml each); peptide=CD31peptide 551-574 (100 μM).

FIGS. 4 and 5 illustrate the principle of the method of according to theinvention. On FIG. 5, the black box represents a discriminatingantibody. The white box represents a capture or a signaling antibody.

FIG. 6A shows that shed CD31 can be used as a diagnostic and/orprognostic marker for inflammatory diseases. White boxes are indicativeof a bad prognostic of the patient, and black boxes are indicative of agood prognostic of the patient. “VS” stands for the erythrocytesedimentation rate. “CRP” stands for the C-reactive protein. “sCD31Total” stands for the total level of soluble forms of CD31, measuredwith the method of the invention. “sCD31^(clivé)” stands for the shedextodomain of CD31, measured with the method of the invention.

FIG. 6B shows that the method of the invention can be used for measuringshed ectodomains of GPVI.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 corresponds to the sequence of human CD31.

SEQ ID NOs: 2 and 3 correspond to CD31 peptides.

SEQ ID NO: 4 corresponds to a scramble peptide used as a negativecontrol.

EXAMPLES Example 1 Material and Methods

Assessment of CD31⁺ and CD31^(shed) blood leukocytes. Ten-color flowcytometry was performed on peripheral blood leukocytes from 5 healthyindividuals either in basal conditions or after overnight stimulationwith soluble 1 μg/ml of purified anti-CD3 antibody (R&D Systems).Ten-color flow cytometry was performed after erythrocyte hypotonic lysis(10 minutes at 37° C. 1:10 v:v in Tris 10 mM, NH₄Cl 155 mM, KHCO₃ 10 mM,pH 7.4) on heparinized peripheral blood leukocytes from 5 healthyindividuals, fixed in PBS/Formaldehyde 1%/FCS 1% for 4 minutes at 37° C.prior to processing. All experiments on human blood were approved by theInternational Ethical committee (see world wide web pageclinicaltrials.gov; Identifier: NCT00430820). Pelleted cells wereincubated for 30 minutes at room temperature and protected from lightwith a cocktail of fluorescent monoclonal antibodies directed to CD3(PE-Texas Red), CD4 (PE-Cy7), CD8 (PerCP), HLA-DR (APC-Cy7), CD45RA(Pacific Blue), and CD31 (WM59, PE) from BD Biosciences and anti-CD20(AlexaFluor®700) and anti-CD31 (PECAM 1.2, FITC) from Invitrogen (1 μlof each). At least 50,000 events were acquired in the lymphocyte gateusing a BD LSRII® equipped with 3 lasers (405, 488 and 633 nm) andanalysed with BD DIVA® 6.0 software.

Subtractive measurement of soluble CD31. To detect the splice variantand truncated CD31 in plasma and the culture supernatant, a cytokinebead array (CBA®, BD) has been customized. Three differently functionalCBA beads (A9, D5 and E9) were coupled with either one of the followingpurified monoclonal anti-CD31 antibodies JC70A (domain 1, DAKO), MEM-05(domain 5, Zymed) and PECAM 1.2 (domain 6, Invitrogen). The coupledbeads were then incubated with the plasma of the same 5 healthy controlsor the culture supernatant and positive binding of circulating CD31 wasdetected by a fourth anti-CD31 monoclonal antibody, WM-59 (domains 1-2)coupled to PE (BD). The concentration of plasma CD31 including at leastdomain 1 (JC70A), or domains 1 to 5 (MEM-05) or all the extracellulardomains 1 to 6 of CD31 (PECAM 1.2) was determined by analysing themedian fluorescent intensity of the detecting antibody on 1000 gatedbeads on samples and serial dilutions of the same standard (recombinant,full length extracellular CD31, R&D Systems). The standard curve wasobtained for each of the beads using the same known concentrations ofthe recombinant CD31 in order to overcome any bias due to differences inbinding affinity of the diverse antibodies. The concentration in ng/mlof CD31 determined with PECAM 1.2 coupled beads (dom1-6) was subtractedfrom the one obtained using MEM-05 coupled beads to obtain the amount ofcirculating CD31 lacking dom6 (dom 1-5). The latter was subtracted fromthe concentration of CD31 obtained using the JC70A-coupled beads tocalculate the value of soluble CD31 lacking both dom 5 and 6 butcontaining at least domains 1 and 2 (dom 1-2).

Assessment of CD31-ITIM phosphorylation. Log-phase Jurkat cells (10⁷cells/condition) were either left unstimulated (negative control) orincubated with pervanadate (positive control) or stimulated withanti-CD3 and anti-CD28 antibodies (R&D Systems, 1 μg/ml each) in thepresence or absence of peptide 551-574 (100 μM), or incubated with thepeptide alone during 20 minutes. Cells were then lysed with 1 ml of RIPAbuffer on ice for 30 minutes, ultracentrifuged and 16 μl of thesupernatant was incubated with PECAM 1.2-coated Functional E9 CBA® beads(BD) for 2 hours at room temperature. Beads were subsequently washedwith CBA washing buffer and incubated with 2 μl of undiluted rabbitanti-CD31 phospho-tyrosine 686 (pY686) sera followed by two washings andincubation with AlexaFluor®488-conjugated (Fab′)₂ fragments (1:100 inCBA washing buffer) of goat-anti-rabbit IgG (Invitrogen). The beads(2000/condition) were finally analysed by flow cytometry in the FITCchannel (530/30 nm) and data are expressed as Median fluorescenceintensity (MFI)±the percentage of the coefficient of variability (% CV)calculated with the DIVA 6.0® software (BD). Duplicate lysate aliquotsand serial dilutions of recombinant CD31 were incubated with the PECAM1.2-coated beads and the amount of dom1+ cell-bound CD31 was revealedusing anti-CD31 VM59-R-PE (dom1) and PECAM 1.2-FITC (dom6) antibodies.

Fluorescent peptide binding. For visualisation of peptide binding toCD31⁺ and CD31^(shed) CD4⁺ T cells, freshly purified peripheral bloodleukocytes prepared as above were washed with a buffered solutioncontaining 2 mM EDTA (to avoid endocytosis of the peptide) and incubatedovernight at room temperature in a dark humidified chamber with 50 μMFITC-labelled CD31 peptide 551-574 and 1:10 dilution of fluorescentmonoclonal anti-CD31 (PE) and anti-CD4 (APC) antibodies (BD Biosciences)in a poly-D-Lysine coated Ibidi® 8-well culture chamber (Biovalley).Cells were then washed twice, nuclei counterstained with DAPI anddigital images of a 0.3 μm intracellular section were acquired on aZeiss Axiovert M200 microscope (×63 immersion objective) equipped withthe ApoTome® and a cooled monochromatic digital camera (Zeiss).

Calcium mobilization assay. Spleen cells from C57BI/6 mice were preparedas described in Caligiuri et al. (2005 Arterioscler Thromb Vasc Biot25:1659-1664). Cells were incubated with Fluo-3AM (Invitrogen, # F1242)as per the instructions of the manufacturer. Fluorescence ofcalcium-bound tracer was measured in the FITC channel on an LSRII®cytometer (BD Biosciences) prior to and during 60 seconds following theaddition of hamster anti-mouse CD3/CD28 monoclonal antibodies (40 μg/mleach) and rat/hamster compBead® (1:50) either alone or in the presenceof rat anti-mouse CD31 antibody (clone 390, 10 μg/ml) or in the presenceof CD31 peptide 551-574 (100 μM). Negative controls included rat IgGisotype control and scramble peptide. Antibodies and compBeads® werefrom BD Biosciences.

Plasmon Surface Resonance. Homophilic binding association anddissociation constants were calculated by surface plasmon resonance(BIAcore® 2000, GE). In brief, peptide 551-574 was coated at 3400resonance units (RU) on CM5 chips according to the manufacturer'sinstructions. Soluble peptide 551-574 (12.5, 25, 50 and 100 μM in 200 μlof 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween 20) was injected at 20μl/min at 25° C., on the peptide-coated channel and on an uncoatedchannel. Dissociation was monitored for 300 seconds. Association (kon)and dissociation (koff) constants were calculated using theBIAevaluation® 3.0 Software (GE). Injection of peptide 551-574 on achannel coated with the scramble peptide yielded negligible signal.

Evaluation of immunoregulation in vitro. CD8⁺ T cell-mediated cytolyticactivity against allogeneic mouse aortic smooth muscle cells andmeasurement of macrophage gelatinase (MMP-2/9) activity were performedas previously described for human cells in Caligiuri et al. (2006Arterioscler Thromb Vasc Biol 26:618-623) using kits and reagents fromInvitrogen. Briefly, primary cultures of FVB/N mouse aorta smooth musclecells were labelled with the lipophylic tracer DIO (green) andco-cultured for 3 hours with CD8+ T cell-enriched spleen cells fromC57BI/6 mice (n=3 scramble peptide and n=3 peptide 551-574, 50 μM).Cytolysis was evaluated by intracellular accumulation of propidiumiodide (PI). Cells were analysed by flow cytometry and the % ofcytolysis was calculated by expressing the number of dead (PI+) cellsamong the target (DIO+) cells. Intracellular MMP-2/9 (gelatinase)activity was measured by flow cytometry in 7-day bone-marrow derivedmacrophages from C57BI/6 mice (n=3 scramble peptide and n=3 peptide551-574, 50 μM) three hours after the incorporation of OregonGreen®gelatine (MFI). T-cell proliferation was performed using either humanperipheral blood mononuclear cells of spleen cells from C57BI/6(CD31^(+/)+) and CD31^(−/−) mice (Charles River France) as previouslydescribed (Caligiuri et al. Arterioscler Thromb Vasc Biol 25:1659-1664).Briefly, cells were plated in triplicates at 0.2×10⁶ cells/well in a Ubottom 96-well plate in complete medium (RPMI 1640, 1% pyruvate, 1%glutamine, 1% penicillin-streptomicyne-fungizone, 10% decomplementedfetal calf serum, all from Invitrogen) containing 1 μg/ml anti-mouseCD3/CD28 or 5 μg/ml anti-human CD3 antibodies (BD) as appropriate. CD31(551-574) and scramble peptide at 25, 50 and 100 μM final concentrationwere deposited in the wells just before cell plating. Plated cells werecultured for 72 hours in 5% CO₂ at 37° C. (³H) thymidine (0.5 μCi/well)was added for the last 16 hours and proliferation evaluated using aTomtec harvester and analysis on a Wallac micro beta counter. Data areexpressed as mean±SEM of cpm in triplicates.

Evaluation of immunoregulation in vivo. Delayed type hypersensitivity(DTH) suppression was evaluated as described in the “Current Protocolsin Immunology (2001) 4.0.1-4.0.2 Unit 4.2”. Briefly, TNCB(2-chloro-1,3,5-trinitrobenzene, Fluka #79874) was dissolved inacetone/olive oil (1:1 v/v) at a concentration of 10 mg/ml. BALB/c mice(n=6/group) were primed by painting the shaved regions of the abdomen awith a total 0.2 ml of the preparation (n=6/group). The experimentincluded 3 groups for peptide 551-574 (10, 50, 100 μM) and 1 grouptreated with scramble peptide at 100 μM). Five days after priming, 10 μlof the TNCB-solvent mixture was painted on the right pinna, 30 minutesafter subcutaneous (interscapolar) administration of the peptide 551-574or the scramble peptide. Ear thickness increases were calculated bysubtracting the thickness of the right and the left pinna of each mouse(right-left/left×100), measured at 24 h with a dial caliper (“Pocotest”,Kroeplin Langenmesstechnick). The measure was performed 5 times on eachear and averaged for further analysis. The immunosuppressive effect ofthe peptide was calculated as % suppression=(1-ΔTE/ΔTS)×100, whereΔT=(ear thickness 24 hr after elicitation)−(baseline ear thickness),E=sensitised animals, S=treated animals. Data are expressed as mean±SEM.

Detection of atherosclerotic lesion size and aneurysm formation. Male28-week old apolipoprotein E^(−/−) mice (n=8-10 mice/group) from ourbreeding facility were maintained on a regular chow diet and kept understandard conditions. Acceleration of atherosclerosis and aneurysmformation was induced by subcutaneous angiotensin II (Sigma, #A9525)infusion (1 mg/kg/d) for 28 days using osmotic minipumps (Alzet, #2004)as previously described (Daugherty et al. J Clin Invest 105:1605-1612).All experiments were approved by our institutional Ethical committee.Atherosclerotic lesions size were measured as previously described(Caligiuri et al. Arterioscler Thromb Vasc Biol 25:1659-1664). Theseexperiments were repeated twice with similar results.

Peptides. All experiments on human material were carried out using thehuman peptide sequence while the mouse equivalent was used in all mouseexperiments. The sequences of the peptides are shown in the table below.

SEQ ID NO: 2 Human NH2 - NHASSVPRSKILTVRVILAPWKK - COOH SEQ ID NO: 3Mouse NH2 - SSMRTSPRSSTLAVRVFLAPWKK - COOH SEQ ID NO: 4 Scramble NH2 - SMPAVRSRFSATSLVTLKSRWPK - COOH

Example 2 The Apparent Loss of CD31 at the Surface of Blood Lymphocytesis Due to its Shedding Between the 5th and 6th Extracellular Ig-LikeDomains

In order to establish whether the loss CD31 was restricted to part orextended to the totality of its 6 extracellular Ig-like domains, amulticolor flow cytometry analysis of whole blood leukocytes from 5healthy donors using two different antibodies specifically recognizingthe membrane-distal and membrane-proximal Ig-like domains of themolecule was performed. To be able to discriminate between the differentleukocyte populations and assess their state of maturation andactivation, a panel of lineage markers as well as the expression ofCD45RA and HLA-DR were simultaneously used. While the expression ofCD31, as detected by a monoclonal antibody specific for the firstdomains of CD31 (clone WM-59, dom1-2) was recognized on naïve but not onactivated/memory blood T cells, all cells expressed themembrane-proximal extracellular fragment of the molecule detected byanother monoclonal antibody specific for the 6^(th) Ig-like domain ofCD31 (clone PECAM 1.2, dom6), irrespective of their state ofmaturation/activation (FIG. 1).

Flow cell cytometry showed that T-cell receptor (TCR) engagement inducesa shift of >80% of blood resting T cells from a CD31 dom1⁺/dom6⁺ to adom1⁻/dom6⁺ (CD31^(Shed))phenotype. Molecular analysis of the membraneproteins from cultured T-cell lysates demonstrated that >99% of the Tcell-bound CD31 molecules drop the distal portion containing dom1already 5′ minutes after TCR stimulation in vitro (FIG. 2 a). Analysisof the supernatant showed that, simultaneously, a single truncatedsoluble protein limited to the first 5 Ig-like domains of CD31,accumulates in the culture supernatant (FIG. 2 b). Furthermore, theanalysis of the plasma of the same healthy donors showed that major partof soluble CD31 in plasma was constituted of a truncated moleculecomprising Ig-like domains 1 to 5 and specifically lacking themembrane-proximal 6^(th) domain (FIG. 2 b) that always remains anchoredto the apparently CD31-negative (CD31 dom1⁻) lymphocytes both in vitroand in vivo. Only a minimal fraction of soluble CD31 contained all 6extracellular domains predicted in the previously reported (Goldbergeret al. J Biol Chem 269:17183-17191) variant spliced form both in culturesupernatant and in plasma (FIG. 2 b). No significant other cleavage ofthe molecule occurs upstream of the 5^(th) domain since the latter wasvirtually always present concomitantly with the first domain in thetruncated soluble CD31 proteins (FIG. 2 b).

Here it is demonstrated that the assumed loss of the molecule onactivated/memory T lymphocytes is actually incomplete and results fromshedding of CD31 between the 5th and 6th extracellular Ig-like domains.CD31 shedding occurred immediately after cell activation on Tlymphocytes and was accompanied by the accumulation of the truncatedmolecule in the supernatant together with trace levels of the splicedvariant produced by the cells. This finding was unsuspected because allcommercially available tests to detect plasma CD31 use antibodiesdirected to CD31 domains 1 to 5, and therefore cannot discriminatebetween the spliced variant (containing all the 6 extracellular domains)and the truncated (domains 1 to 5) forms of CD31. The subtractiveimmunosorbent assay described herein is able to discriminate between thetwo forms of soluble CD31 and precisely quantify the proportion of eachof them in the plasma. This assay showed that the major part of plasmaCD31 comprises domains 1 to 5 but lacks the membrane-proximal 6thdomain, which remains anchored to blood CD31 dom1-lymphocytes.Therefore, it is proposed to refer to these lymphocytes as CD31 shedrather that CD31 “negative” cells. Previous work in vitro had indicatedthat CD31 shedding at an unidentified position N-terminal from thetransmembrane segment of the molecule can occur in endothelial cellsundergoing apoptosis (Ilan et al. 2001. Faseb J 15:362-372). For thefirst time, it is shown herein that shedding is responsible for the CD31(incomplete) loss on blood lymphocytes and that the circulating CD31consists mainly of a truncated form derived from its cleavage betweenthe Ig-like domains 5 and 6, rather than of the secreted spliced variantform. Genetic polymorphisms for CD31 have been described, but thepredictive value of soluble CD31 levels was conflicting either inatherothrombosis or other dysimmune diseases. In fact, while the amountof the spliced form can be predicted by different genetic variants, theproportion of the form resulting from protein shedding is not determinedby CD31 gene polymorphism. It is proposed that the disparity between thedifferent studies was due to fact that circulating CD31 is a mixture ofthe genetic variant and of the truncated form and discrimination betweenthe two forms of CD31 was not possible. The subtractive method describedherein will allow the differentiation of the prognostic value determinedby genetic variants of CD31 independently of that linked to CD31shedding.

Example 3 A Peptide Contained in the Residual Extracellular CD31Fragment on CD31^(shed) T Cells Enhances Phosphorylation of CD31-ITIM

A CD31 dom6-derived synthetic peptide corresponding to thejuxta-membrane 23 aminoacids (551-574) of the ectodomain of the humanmolecule binds both to CD31 dom1⁺ and to CD31 dom1⁻ (CD31^(shed)) CD4⁺ Tlymphocytes ex vivo. Importantly, the binding of this peptide on T cellshas functional consequences on immune cell control since it exerteddose-dependent inhibition of human peripheral blood T-cell proliferationin vitro (FIG. 3 a). To assess whether the inhibitory effect of thepeptide could be mediated by homophilic binding and engagement of theCD31 signaling, the level of phosphorylation of the CD31 ITIM tyrosineat position 686 (₆₈₆ITIM) in cultured T cells was evaluated. Stimulationof the TCR by anti-CD3 and anti-CD28 antibodies alone or the solepresence of the peptide induced a minor increase of CD31 pY686 (FIG. 3b) but concomitant TCR-stimulation in the presence of the peptideboosted the phosphorylation the CD31₆₈₆TIM by a factor of >23 (FIG. 3b).

Example 4 Detection of Shed Cd31 in Plasma from Patients Suffering fromAtherothrombosis and in Unaffected Individuals

The total amount of CD31, the amount of shed CD31 and the amount ofspliced CD31 has been measured both in eleven individuals suffering fromatherothrombosis and in twenty-three unaffected individuals.

The group “Atherothrombosis” comprised eleven individuals suffering fromchest pain even at rest and presenting an abnormal coronarography.

The group “No Atherothrombosis” comprised twenty-three individuals. Asub-analysis was carried out on the group “No Atherothrombosis”, whichwas found to comprise:

-   -   eight individuals presenting a normal coronarography and a        normal carotid echodoppler in spite of chest pain;    -   four individuals presenting a normal coronarography in spite of        chest pain, but in whom atherosclerosis was detected by carotid        echodoppler; and    -   eleven individuals suffering from chest pain only on effort and        presenting an abnormal coronarography (i.e. suffering from        coronary atherosclerosis without thrombosis).

The total amount of CD31, the amount of shed CD31 and the amount ofspliced CD31 was determined as follows.

1. The total amount (1 μl/test) of beads (E9, coupled with clone JC70A,DAKO) was transferred to a conical tube and centrifuged at 200 g for 5minutes. The supernatant was carefully discarded and replaced with sameamount of serum enhancement buffer (BD #51-9002150), and incubated atroom temperature for 15 minutes.

2. The fluorescently-labeled antibody mix (PE-WM59; FITC-HC1/6;PB-PECAM1.2) was prepared, each at 1 μg/ml, 1 μl each/condition.

3. 1 tube precondition was prepared, each containing 3 μl of a standarddilution or a plasma sample. The reconstituted beads were centrifuged at200 g for 5 minutes, the supernatant was discarded and the serumenhancement buffer was replaced with the fluorescently-labeled antibodymix. 3 μl of this solution was distributed in each of the tubescontaining the standard dilution and samples, and the solution incubatedfor 1 hour at 4° C. in the dark.

4. 150 μl of Washing buffer (BD # 51-9003797) were added to each tube,and the signal was acquired.

As shown in the table below, the percentage of shed CD31 was higher inindividuals suffering from atherothrombosis than in unaffectedindividuals, in spite of the fact that all individuals were sufferingfrom chest pain.

CD31 Plasma Level (ng/ml) total splice shed Atherothrombosis 11.55 ±0.7  −7.02 ± 2.69 18.57 ± 2.67 (n = 11) No Atherothrombosis 11.58 ± 0.49 5.26 ± 1.850  6.31 ± 1.85 (N = 23) T-test Prob > F 0.9756 0.0007 0.0006

Total CD31 amounts were similar in the four groups, while the amount ofshed CD31 and the amount of spliced CD31 were significantly different ineach paired group comparison. Shed CD31 was increased in individualswith abnormal coronarography, with highest values in those sufferingfrom atherothrombosis. Splice CD31 was still present in patientssuffering from atherosclerosis without atherothrombosis, while it wasalmost undetectable in patients suffering from atherothrombosis.

These results demonstrate that high levels of CD31 soluble splicevariants associated with low levels of shed CD31 indicates that thepatient suffers from non specific chest pain, eventually associated withcarotid plaques. A slight increase of shed CD31 levels associated withnormal or reduced levels of CD31 soluble splice variants indicates thatthe patient suffers from atherosclerosis. An important increase of shedCD31 levels associated with undetectable amounts of CD31 soluble splicevariants indicates that the patient suffers from atherothrombosis.

Example 5 Use of CBA® Beads for Quantitative Assessment of ProteinAssociation and of Phosphorylation

8.1. Protocol Used for Assessing the CD31 ITIM-Dependent InhibitoryPathway in T Cells

Lysis of the cells. Jurkat T cells in log phase (10×10⁶/ml, 1 ml) wereeither left untouched (negative control) or incubated with Na₃VO₄/H₂O₂(sodium ortovanadate which is a tyrosin phosphatase inhibitor, positivecontrol for tyrosin phosphorylation). In parallel, cells were stimulatedeither with CD3 or with CD3+ CD31 (domain 2, WM-59 antibody) byantibody-mediated crosslinking. After 20′ incubation at 37° C. in 5%CO₂, the cells were lysed on ice for 30′ with a RIPA buffer containing acocktail of protease and phosphatase inhibitors. Lysates wereultragentrifuged and supernatants were used either straightforward oraliquoted and stored at −20° for further analysis.

Capture of the target protein on a solid support (1 hour at roomtemperature). This step follows the principle of immunoprecipitation butthe signaling complex is nor denatured neither reduced, and noelectrophoresis and/or blotting is carried out. As a solid support, CBA®functional beads previously coupled with an antibody directed to themembrane-proximal CD31 Ig-like domain 6 (clone MBC 78.2 also calledPECAM1.2) were used following the manufacturer's instructions. Analiquot of 20 μl of lysate supernatant from each condition was incubatedwith 5 μl PECAM1.2-CBA® for 1 hour at room temperature.

Detection of molecules associated with CD31 and of the phosphorylationstate of CD31 ITIM 686. After washing of the beads once with CBA washingbuffer (100 μl/tube), the beads were been aliquoted in 4 separate tubesand incubated with fluorescent antibodies directed to:

-   -   CD3-FITC;    -   CD28-PE;    -   CD31 domain 6 (PECAM1.2 FITC)    -   CD31 domain 2 (WM-59 PE)    -   phosphoTyrosin (clone 4G10 FITC);    -   phosphoSerin/Threonin (labelled with FITC)    -   CD31 phosphotyrosin 686 (rabbit polyclonal conjugated with        alexafluor 488 antirabbit secondary antibody); or    -   SHP2-PE.

After 1 hour incubation at room temperature in the dark, beads werewashed once with 100 μl CBA washing buffer and more than 600 beads wereacquired using an LSR II and DIVA® software.

4. Analysis. The median fluorescence in each fluorescent channel wasrecorded. The DIVA® software also calculated the % CV (variabilitycoefficient), which can be considered as an equivalent of error barvalue for repeated measure and allow statistical comparison betweensamples.

8.2. Results

The results are shown in the tables below.

(ng/ml) (ng/ml) % MFI MFI CD31 CD31 shed pY pSer/ dom6 dom2 CD31 (4G10)pThreo Negative unstimulated  19 18 5  485 284 control PositiveNa3VO4/H2O2  103 77 25 1205 324 pY control T-cell CD3 crosslink 9080 8399 2681 130 activation Antibody CD3 + CD31 2389 46 98 2312 267 CD31crosslink treatment Peptide CD3 10962 ¹    67 ² 99   3277 ³ 329 CD31crosslink + treatment CD31 peptide

MFI CD31 MFI MFI MFI pY686 SHP-2 CD3 CD28 Negative control 1578 165 197 8165 Positive pY control 10667  193 354  3932 T-cell activation 4198 90 17876  25169 Antibody CD31 2268 149 2425  17147 treatment PeptideCD31 96632 ³    185 ⁴ 20096 ⁵     24306 ⁵ treatment

A value in bold indicates an increase versus CD3 crosslink conditions,and values in italic indicate a decrease.

¹Engaged CD31 molecules oligomerize in cis. CD31 molecule beingphysiologically engaged as a regulatory molecule in T-cell activation,this explains the increase of CD31 domain 6 amount in T-cell stimulatedsamples.

²If only domain 2 of CD31 was detected, one would conclude that theamount of the captured molecule was similar in all samples. Indeed, mostof CD31 cis-oligomerized molecules are shed between domain 6 and 2 uponT-cell activation as show in the column “% shed”.

³Total phosphoTyrosin is slightly decreased by CD31 antibody treatmentwhile it is slightly increased by CD31 peptide treatment. Indeed, notonly CD31 ITIMs can be phosphorylated, but also tyrosins present onother membrane proteins (e.g. CD3 and CD28) which are associated withCD31 upon T-cell activation. The differences specific for CD31phospholTIMs could detected by using antibodies directed tosequence-specific phospholTIM. Not only phosphotyrosine are increased,but also phosphoserine/threonine. These aminoacid can be phosphorylatedeither in the CD31 cytoplasmic tail, or in one of the associatedmolecule.

⁴The amount of SHP-2 associated to CD31 is increased by 50% with theantibody treatment and by 100% with the peptide treatment.

⁵The amount of activating receptor (CD3) and the co-stimulatory moleculeassociated to the CD31 (CD28) are decreased by the antibody treatment,while they are increased by the CD31 peptide. This can be viewed as acounter-regulatory mechanism of the cell in the view of the dramaticincrease of CD31 ITIM phosphorylation that counteracts T-cellactivation.

8.3. Conclusion

All these data were acquired starting from a very small amount of thesame sample (20 μl). The use of multicolor flow-cytometry warrants theiranalysis in a simultaneous way. The availability of a standard allowsexact determination of absolute amounts of each molecule in thesignaling complex.

The above experiment was performed using either FITC (FL1) or PE (FL2)conjugated antibodies. The use of non-fluorescent beads for proteincapture allows the use of at least two other fluorophores for use in theblue laser (PerCP or PE-Cy7 or PE-Cy5, FL3 and APC or Cy5, FL4). Theavailability of additional lasers (red, violet, uv) on the cytometerfurther expands the capacity of this method. Up to 17 differentfluorescent antibodies can be used simultaneously and therefore up to 17different parameters (associated membrane protein, signaling molecules,phosphorylated sequences) can be detected simultaneously on the samesample, with a powerful statistical value due to the high number ofbeads that can be acquired.

The above method has numerous advantages compared to prior art methods.The table below compares this method with co-IP/WB, CBAFIex andCASE/Phosphlow.

Method Phoflow/ according to Operation IP/WB CBAFlex CASE the inventionProtein extraction 30 min 30 min N/A 30 min Immunoprecipitation 1-12 hN/A N/A N/A SDS-PAGE 2 h N/A N/A N/A Blot 2 h N/A N/A N/A Fixation N/AN/A 1 h N/A Blocking 1 h N/A 1 h N/A Primary Antibody 1-18 h 1 h 1 h 1 hWashing 30 min 5 min 15 min 0-5 min Secondary Antibody 1 h N/A 1 h N/AWashing 30 min N/A 15 min N/A Detection develop. 60 min N/A 15 min N/AData Acquisition 1 h <10 sec/samp 15 min <10 sec/samp Multi-target N/Aup to 20 ≧2 up to 20 Time ~12-36 h ~1.5 h ~7 h ~1.5 h Sample size ≧100μL 20-50 μl ≧50 μL ≦5 μl Specific interactions Yes (1) N/A N/A Yes (≧1)

Compared to co-IP/WP, this method is quantitative, direct and much morerapid. In addition, it allows the simultaneous detection of the targetprotein, of phosphomolecules and of associated membrane andintracellular molecules. It also allows the simultaneous analysis ofmuch more parameters since a Western Blot can only be rehybridized alimited number of times, whereas up to 20 antibodies can be usedsimultaneously in the frame of this method with the most recentgeneration of cytometers.

Compared to CASE/Phosphlow, or CBA Flex set, this method is much morespecific since the target molecule is captured and the interactionswithin the molecular complex can be analyzed. In addition, it allows thesimultaneous detection of the target protein, of phosphomolecules and ofassociated membrane and intracellular molecules.

Example 6 Optimization of the Protocol

5 μl of sample are incubated for 30-60′ at 4° C. in the dark with 1 μlof a mix containing functional CBA beads-immobilized monoclonal MEM-05antibody (CD31 domain 5, Exbio, Caltag), WM-59 (CD31 domain 2) and PECAM1.2 (also called MBC 78.2, CD31 domain 6) coupled to a fluorophore.After incubation, beads are diluted with 200 μl of assay diluent bufferand acquired. The median fluorescent intensity of the PE and FITCchannel on more than 1000 gated beads is analyzed on samples and serialdilutions of the standard (recombinant, full length extracellular CD31,R&D Systems). Two standard curves are obtained with each of thedetecting antibodies simultaneously used with recombinant CD31 in orderto overcome any bias due to differences in binding affinity of thediverse antibodies. With three discriminating antibodies directed todomains 2, 5 and 6, it is possible to discriminate full CD31 from CD31lacking either domain 6 (Δ6) from CD31 lacking both domain 5 and 6(Δ5-6).

To increase the specificity of the test, monoclonal antibodies directedto CD31 domain 1 (clone JC70A, Dako) are coupled to capture beads.Detection is based on three fluorescent antibodies directed to CD31domain 2 (WM-59, BD), domain 5 (HC1/6, Biosource) and domain 6. PurifiedPECAM 1.2/MBC 78.2 is coupled with a suitable fluorophore. With currentfunctional beads, an optimal set of fluorophores is FITC, PE and PerCP.This combination allows measurement on any basic blue-laser cytometer(FACScan, FACSCalibur, etc) available in hospital biology laboratory.

With FITC and PE as fluorophores (see example 5), the lowest detectionlimit is 3 ng/ml. This results in negative values of spliced CD31 inpatients presenting a high risk of suffering from atherothrombosis. Incontrast to this, different fluorophores may lead to an increasedsensitivity. Therefore, different combinations of fluorophores and ofantibodies are tested.

Example 7 Analysis of the BIOcore Cohort

A cohort of patient has been analyzed. The individuals were classifiedin different groups, as set forth in Example 4.

The cell analysis by cytometry confirmed that the percentage of T (CD3+)lymphocytes that display a truncated (shed) CD31 is significantly higherin patients at risk of acute coronary events (see Table 1).

TABLE 1 Oneway analysis of CD31shed (% of CD3 cells) by Group Level -Level Difference Lower CL Upper CL p-Value ACS Norm 16.06119 10.739521.38292 <0.0001* ACS Carotide 15.99231 9.6004 22.38419 <0.0001* ACS SA12.79007 7.9876 17.59257 <0.0001* SA Norm 3.27111 −2.2928 8.83498 0.2462SA Carotide 3.20223 −3.3926 9.797.9 0.3377 Carotide Norm 0.06888 −6.91327.05092 0.9844 In the above table, “ACS” stands for acute coronarysyndromes, “Carotide” stands for peripheral atherosclerosis, “Norm”stands for normal coronary angiogram, and “SA” stands for stable angina.

Subpopulation analysis showed that this conclusion is valid both forCD4+ T (CD3+) cells and for CD8+ T (CD3+) cells (see Tables 2 and 3,respectively).

TABLE 2 Oneway analysis of CD31shed (% of CD4 cells) by Group Level -Level Difference Lower CL Upper CL p-Value ACS Carotide 26.37179 18.205034.53856 <0.0001* ACS Norm 19.24452 12.4451 26.04397 <0.0001* ACS SA16.43631 10.3003 22.57235 <0.0001* SA Carotide 9.93548 1.5094 18.361570.0213* Norm Carotide 7.12727 −1.7935 16.04806 0.1161 SA Norm 2.80821−4.3006 9.91704 0.4351

TABLE 3 Oneway analysis of CD31shed (% of CD8 cells) by Group Level -Level Difference Lower CL Upper CL p-Value ACS Carotide 8.364103 2.1095414.61867 0.0093* ACS Norm 6.664452 1.45706 11.87185 0.0126* ACS SA4.435567 −0.26376 9.13489 0.0640 SA Carotide 3.928536 −2.52463 10.381710.2300 SA Norm 2.228886 −3.21545 7.67322 0.4186 Carotide Carotide1.699650 −5.13239 8.53169 0.6227

Plasma analysis also confirmed that the level of total soluble CD31 isnot able to discriminate between the groups (see Table 4).

TABLE 4 Oneway analysis of total soluble CD31 (ng/ml) by Group Level -Level Difference Lower CL Upper CL p-Value Norm ACS 1.017336 −0.717482.752148 0.2474 Norm SA 0.965691 −0.84806 2.779439 0.2934 Norm Carotide0.825823 −1.45023 3.101875 0.4733 Carotide ACS 0.191513 −1.892162.275182 0.8557 Carotide SA 0.139868 −2.00997 2.289702 0.8976 SA ACS0.051645 −1.51391 1.617195 0.9480

Only the method according to the invention, which allows measuringspliced CD31 and shed CD31 in plasma, can be employed for diagnosingand/or prognosing acute coronary syndromes (see Tables 5 and 6,respectively).

TABLE 5 Oneway analysis of spliced CD31 (ng/ml) by Group Level - LevelDifference Lower CL Upper CL p-Value Norm ACS 4.723988 1.97055 7.4774280.0010* SA ACS 3.288827 0.80403 5.773619 0.0100* Carotide ACS 3.255752−0.05138 6.562888 0.0536 Norm Carotide 1.468236 −2.14424 5.080714 0.4220Norm SA 1.435162 −1.44356 4.313886 0.3250 SA Carotide 0.033074 −3.379083.445224 0.9847

TABLE 6 Oneway analysis of shed CD31 (ng/ml) by Group Level - LevelDifference Lower CL Upper CL p-Value ACS Norm 3.707532 0.87674 6.5383210.0108* ACS SA 3.238062 0.68347 5.792657 0.0135* ACS Carotide 3.065120−0.33492 6.465159 0.0767* Carotide Norm 0.642412 −3.07155 4.3563730.7322 SA Norm 0.469470 −2.49012 3.429064 0.7537 Carotide SA 0.172942−3.33506 3.680946 0.9223

In particular, the proportion of shed soluble CD31 (% of total) is veryuseful for distinguishing between acute coronary syndrome, stablecoronary disease and peripheral atherosclerosis (see Table 7).

TABLE 7 Oneway analysis of shed CD31 (% of Total soluble forms of CD31)by Group Level - Level Difference Lower CL Upper CL p-Value ACS Norm22.00424 8.4993 35.50915 0.0017* ACS Carotide 18.24801 2.0274 34.468650.0278* ACS SA 18.06073 5.8735 30.24799 0.0041* SA Norm 3.94351 −10.175918.06290 0.5808 Carotide Norm 3.75623 −13.9620 21.47449 0.6750 SACarotide 0.18729 −16.5484 16.92299 0.9823

Importantly, it was also found that the measure in the plasma ofpatients of spliced and of shed CD31, and not that of total CD31, allowspredicting recurrence of Major Adverse Cardiovascular Events (MACE, suchas death, fatal and non fatal myocardial infarction).

TABLE 8 1-way Test, ChiSquare Approximation Oneway analysis of:ChiSquare DF Prob > ChiSq Total CD31 (ng/ml) by MACE 0.1564 1 0.6925Spliced CD31 (ng/ml) by MACE 5.0064 1 0.0253* Shed CD31 (ng/ml) by MACE4.4985 1 0.0339*

Example 8 Shed CD31 as a Diagnostic and/or Prognostic Marker ofInflammatory Diseases

It was investigated whether the measure of soluble spliced and shed CD31using the method of the invention could help evaluating the risk oftreatment failure in patients with chronic inflammatory diseases.

Data from 73 patients affected either by rheumatoid arthritis orspondyloarthritis were collected.

It was found that plasma levels of CD31 were associated both with thepatient's outcome and with a positive response to biotherapy (see FIG.6A). This cannot be predicted by the currently used biomarkers (ESR andCRP).

The above patients suffered from very severe inflammation. In suchcases, the level of shed CD31 may increase of several folds above thebaseline levels (even above 1000 ng/ml), and consequently lead to asignificant increase of total soluble CD31. Therefore, not only shedCD31 but also total soluble CD31 can be used as a biomarker forinflammation when the inflammation is very severe.

However, differentiating shed CD31 form total soluble CD31 is requiredin patients in which the inflammatory state is less obvious.

Example 9 Measure of the Plasma Levels of Soluble GPVI

The method according to the invention was adapted for measuring plasmalevels of soluble GPVI. The soluble form of GPVI is cleaved fromplatelets upon activation and which could evaluate the occurring ofthrombus formation in patients at risk.

A reproducible method for measuring shed GPVI in the plasma using (i) anantibody for capture (coupled to the CBA beads); and (ii) afluorescently labeled natural ligand of GPVI (convulxin) for thedetection has been set up. The antibody for capture was either clone No.3j24.2 or clone No. 9O12.2. These two monoclonal antibodies aredescribed in patent application No. PCT/US2000/018152, published asWO/2001/000810. These antibodies specifically recognize epitopes locatedon the ectodomain of GPVI.

No current ELISA can achieve the sensitivity and precision of this newmethod (see FIG. 6B). Standard curves can be obtained both withrecombinant GPVI dimers and with recombinant GPVI monomers. This findingis valuable since GPVI dimerizes upon platelet activation, just beforebeing cleaved. After shedding it circulates both as a dimer and as amonomer, and the function and/or diagnostic value might differ dependingon the dimerization status.

The method according to the invention thus allows measuring dimers andmonomers shed GPVI in the plasma of individuals.

Specifically detecting the shed form of GPVI can be carried out asfollows. GPVI spliced form comprises the cytoplasmic tail and cantherefore be detected using specific antibodies recognizing thecytoplasmic tail such as those raised against a maltose-binding protein(MBP)-GPVI cytoplasmic tail fusion protein (Suzuki-Inoue et al. J Biol.Chem. 2002 277:21561-6.). On the other hand, the shed form of GPVIcomprises the complete ectodomain. Therefore, the method of theinvention is performed either using specific antibodies (anti-tail andanti ectodomain) on separate beads for the capture and fluorescentlylabeled convulxin for the detection, or vice versa (capture byconvulxin-coupled beads and detection by differently labeled fluorescentanti-tail and anti-ectodomain antibodies).

1. A method for detecting a shed ectodomain of a transmembrane proteinamong soluble forms of said transmembrane protein in a biologicalsample, wherein said soluble forms include a soluble splice variant ofsaid transmembrane protein and optionally said shed ectodomain, whichcomprises the steps of: a) providing a first type of bead linked to anantibody which specifically binds to an epitope located in a region thatis present both on said shed ectodomain and on said splice variant(first discriminating antibody); b) providing at least one second typeof bead linked to an antibody which specifically binds to an epitopelocated in a region that is either present on said shed ectodomain andabsent from said splice variant, or present on said splice variant andabsent from said shed ectodomain (second discriminating antibody); c)providing a fluorescently-labelled ligand which specifically binds to aregion that is present both on said shed ectodomain and on said splicevariant (signaling ligand); d) contacting said antibodies with abiological sample likely to contain said soluble forms of saidtransmembrane protein; e) for each type of bead, measuring the signalobtained with said florescent label by flow cytometry; and f) comparingthe signal obtained for each type of bead; wherein a difference in thesignals measured at step (e) indicates that the biological samplecomprises said shed ectodomain.
 2. A method for detecting a shedectodomain of a transmembrane protein among soluble forms of saidtransmembrane protein in a biological sample, wherein said soluble formsinclude a soluble splice variant of said transmembrane protein andoptionally said shed ectodomain, which comprises the steps of: a)providing a bead linked to a ligand which specifically binds to a regionthat is present both on said shed ectodomain and on said splice variant(capture ligand); b) providing a first type of fluorescently-labelledantibody which specifically binds to an epitope located in a region thatis present both on said shed ectodomain and on said splice variant(first discriminating antibody); c) providing at least one second typeof fluorescently-labelled antibody which specifically binds to anepitope located in a region that is either present on said shedectodomain and absent from said splice variant, or present on saidsplice variant and absent from said shed ectodomain (seconddiscriminating antibody); d) contacting said antibodies with abiological sample likely to contain said soluble forms of saidtransmembrane protein; e) for each fluorescent label, measuring thesignal obtained with said florescent label by flow cytometry; and f)comparing the signal obtained for each fluorescent label. wherein adifference in the signals measured at step (e) indicates that thebiological sample comprises said shed ectodomain.
 3. The methodaccording to claim 1, wherein: i) said soluble forms include at leastthree soluble forms; ii) the discriminating antibodies are chosen insuch a way as to discriminate between said soluble forms; and iii) thecapture ligand or the signaling ligand specifically binds to a regionthat is present on all said soluble forms.
 4. The method according toclaim 1, wherein said capture ligand or signaling ligand is an antibody.5. The method according to claim 1, further comprising the step ofcalculating the percentage and/or the amount of said soluble forms thatcorresponds to said shed ectodomain.
 6. The method according to claim 1,further comprising the step of calculating either the ratio of shedectodomain to soluble forms, or the ratio of soluble splice variant tosoluble forms.
 7. The method according to claim 1, wherein saidtransmembrane protein is CD31.
 8. The method according claim 1, whereinsaid transmembrane protein is glycoprotein VI (GPVI).
 9. The methodaccording to claim 7, wherein the first discriminating antibodyspecifically binds to an epitope located in the first extracellularimmunoglobulin-like domain of CD31, and wherein the seconddiscriminating antibody specifically binds to an epitope located in thesixth extracellular immunoglobulin-like domain of CD31.
 10. The methodaccording to claim 7, wherein: the first discriminating antibodyspecifically binds to an epitope located in the first extracellularimmunoglobulin-like domain of CD31; the second discriminating antibodyspecifically binds to an epitope located in the sixth extracellularimmunoglobulin-like domain of CD31; and the discriminating antibodiesfurther comprise an antibody which specifically binds to an epitopelocated in the fifth extracellular immunoglobulin-like domain of CD31.11. The method according to claim 7, wherein said biological sample isplasma obtained from an individual suffering from or at risk ofsuffering from a thrombotic or an autoimmune disorder.
 12. A method fordiagnosing whether an individual suffers, or is at risk of suffering,from a thrombotic or an autoimmune disorder, which comprises the step ofdetecting a shed ectodomain of CD31 in a biological sample of saidindividual, wherein the presence of said shed ectodomain of CD31indicates that said individual suffers from or is at risk of sufferingfrom said a thrombotic or autoimmune disorder.
 13. The method of claim12, wherein said step of detecting a shed ectodomain of CD31 is repeatedat least at two different points in time in order to monitor theprogression of said disorder in said individual, and/or to assess theseverity of said disorder in said individual, and/or to monitor theresponse of said individual to a drug.
 14. A method for monitoring theresponse of a patient to a drug, said method comprising the steps of: a)detecting shed ectodomains of CD31 in a biological sample of saidpatient before and after onset of a treatment of said patient with saiddrug; b) comparing the levels of shed ectodomains of CD31 detected atstep (a); and, optionally, c) correlating a difference in said levels ofshed ectodomains of CD31 with the effectiveness of the drug for treatingsaid patient.
 15. The method according to claim 12, wherein said step ofdetecting a shed ectodomain of CD31 comprises the steps of: a) providinga first type of bead linked to an antibody which specifically binds toan epitope located in a region that is present both on said shedectodomain of CD31 and on a soluble splice variant of CD31 (firstdiscriminating antibody); b) providing at least one second type of beadlinked to an antibody which specifically binds to an epitope located ina region that is either present on said shed ectodomain of CD31 andabsent from said soluble splice variant of CD31, or present on saidsoluble splice variant of CD31 and absent from said shed ectodomain ofCD31 (second discriminating antibody); c) providing afluorescently-labelled ligand which specifically binds to a region thatis present both on said shed ectodomain of CD31 and on said solublesplice variant of CD31 (signaling ligand); d) contacting said antibodieswith a biological sample likely to contain said soluble forms of saidtransmembrane protein; e) for each type of bead, measuring the signalobtained with said florescent label by flow cytometry; and f) comparingthe signal obtained for each type of bead; wherein a difference in thesignals measured at step (e) indicates that the biological samplecomprises said shed ectodomain of CD31.
 16. A diagnostic kit comprising:i) a first type of bead linked to an antibody that specifically binds toan epitope located in a region that is present both on a shed ectodomainof a transmembrane protein and on a soluble splice variant of saidtransmembrane protein; ii) a second type of bead linked to an antibodythat specifically binds to an epitope located in a region that is eitherpresent on said shed ectodomain and absent from said soluble splicevariant, or present on said soluble splice variant and absent from saidshed ectodomain; and iii) a fluorescently-labelled antibody thatspecifically binds to an epitope located in a region that is presentboth on said shed ectodomain and on soluble said splice variant.
 17. Adiagnostic kit comprising: i) a bead linked to an antibody thatspecifically binds to an epitope located in a region that is presentboth on a shed ectodomain of a transmembrane protein and on a solublesplice variant of said transmembrane protein; ii) a first type offluorescently-labelled antibody that specifically binds to an epitopelocated in a region that is either present on said shed ectodomain andabsent from said soluble splice variant, or present on said solublesplice variant and absent from said shed ectodomain; and iii) a secondtype of fluorescently-labelled antibody that specifically binds to anepitope located in a region that is present both on said shed ectodomainand on soluble said splice variant.
 18. A method for determining whethera candidate molecule is member of a molecular complex which comprisesthe steps of: a) providing a bead linked to an antibody thatspecifically binds to a member of said molecular complex; b) contactingsaid bead with a biological sample containing said molecular complex; c)contacting said beads contacted with the biological sample with at leastone type of fluorescently-labelled antibody that specifically binds tosaid candidate molecule; and d) detecting the fluorescence by flowcytometry; wherein: the detection of a signal at step (d) indicates thatsaid candidate molecule is a member of said molecular complex; and ifmore than one type of fluorescently-labelled antibody is used at step(c), said antibodies are labelled with different fluorescent labels. 19.A kit for analyzing a molecular complex which comprises: i) a beadlinked to an antibody that specifically binds to a member of saidmolecular complex; and ii) at least one type of fluorescently-labelledantibody that specifically binds to a molecule likely to be member ofsaid molecular complex; wherein, if the kit comprises more than onefluorescently-labelled antibody, said antibodies are labelled withdifferent fluorescent labels.
 20. The method of claim 18 or the kit ofclaim 19, wherein said member of a molecular complex is CD31.
 21. Thekit of claim 19, wherein said member of a molecular complex is CD31. 22.The method according to claim 2, wherein: i) said soluble forms includeat least three soluble forms; ii) the discriminating antibodies arechosen in such a way as to discriminate between said soluble forms; andiii) the capture ligand or the signaling ligand specifically binds to aregion that is present on all said soluble forms.
 23. The methodaccording to claim 2, wherein said capture ligand or signaling ligand isan antibody.
 24. The method according to claim 2, further comprising thestep of calculating the percentage and/or the amount of said solubleforms that corresponds to said shed ectodomain.
 25. The method accordingto claim 2, further comprising the step of calculating either the ratioof shed ectodomain to soluble forms, or the ratio of soluble splicevariant to soluble forms.
 26. The method according to claim 2, whereinsaid transmembrane protein is CD31.
 27. The method according to claim 2,wherein said transmembrane protein is glycoprotein VI (GPVI).
 28. Themethod according to claim 26, wherein the first discriminating antibodyspecifically binds to an epitope located in the first extracellularimmunoglobulin-like domain of CD31, and wherein the seconddiscriminating antibody specifically binds to an epitope located in thesixth extracellular immunoglobulin-like domain of CD31.
 29. The methodaccording to claim 26, wherein: a. the first discriminating antibodyspecifically binds to an epitope located in the first extracellularimmunoglobulin-like domain of CD31; b. the second discriminatingantibody specifically binds to an epitope located in the sixthextracellular immunoglobulin-like domain of CD31; and c. thediscriminating antibodies further comprise an antibody whichspecifically binds to an epitope located in the fifth extracellularimmunoglobulin-like domain of CD31.
 30. The method according to claim26, wherein said biological sample is plasma obtained from an individualsuffering from or at risk of suffering from a thrombotic or anautoimmune disorder.
 31. The method according to claim 12, wherein saidstep of detecting a shed ectodomain of CD31 comprises the steps of: a)providing a bead linked to a ligand which specifically binds to a regionthat is present both on said shed ectodomain of CD31 and on a solublesplice variant of CD31 (capture ligand); b) providing a first type offluorescently-labelled antibody which specifically binds to an epitopelocated in a region that is present both on said shed ectodomain of CD31and on said soluble splice variant of CD31 (first discriminatingantibody); c) providing at least one second type offluorescently-labelled antibody which specifically binds to an epitopelocated in a region that is either present on said shed ectodomain ofCD31 and absent from said soluble splice variant of CD31, or present onsaid soluble splice variant of CD31 and absent from said shed ectodomainof CD31 (second discriminating antibody); d) contacting said antibodieswith a biological sample likely to contain said soluble forms of saidtransmembrane protein; e) for each fluorescent label, measuring thesignal obtained with said florescent label by flow cytometry; and f)comparing the signal obtained for each fluorescent label. wherein adifference in the signals measured at step (e) indicates that thebiological sample comprises said shed ectodomain of CD31.
 32. The methodaccording to claim 14, wherein said step of detecting a shed ectodomainof CD31 comprises the steps of: a) providing a first type of bead linkedto an antibody which specifically binds to an epitope located in aregion that is present both on said shed ectodomain of CD31 and on asoluble splice variant of CD31 (first discriminating antibody); b)providing at least one second type of bead linked to an antibody whichspecifically binds to an epitope located in a region that is eitherpresent on said shed ectodomain of CD31 and absent from said solublesplice variant of CD31, or present on said soluble splice variant ofCD31 and absent from said shed ectodomain of CD31 (second discriminatingantibody); c) providing a fluorescently-labelled ligand whichspecifically binds to a region that is present both on said shedectodomain of CD31 and on said soluble splice variant of CD31 (signalingligand); d) contacting said antibodies with a biological sample likelyto contain said soluble forms of said transmembrane protein; e) for eachtype of bead, measuring the signal obtained with said florescent labelby flow cytometry; and f) comparing the signal obtained for each type ofbead; wherein a difference in the signals measured at step (e) indicatesthat the biological sample comprises said shed ectodomain of CD31. 33.The method according to claim 14, wherein said step of detecting a shedectodomain of CD31 comprises the steps of: a) providing a bead linked toa ligand which specifically binds to a region that is present both onsaid shed ectodomain of CD31 and on a soluble splice variant of CD31(capture ligand); b) providing a first type of fluorescently-labelledantibody which specifically binds to an epitope located in a region thatis present both on said shed ectodomain of CD31 and on said solublesplice variant of CD31 (first discriminating antibody); c) providing atleast one second type of fluorescently-labelled antibody whichspecifically binds to an epitope located in a region that is eitherpresent on said shed ectodomain of CD31 and absent from said solublesplice variant of CD31, or present on said soluble splice variant ofCD31 and absent from said shed ectodomain of CD31 (second discriminatingantibody); d) contacting said antibodies with a biological sample likelyto contain said soluble forms of said transmembrane protein; e) for eachfluorescent label, measuring the signal obtained with said florescentlabel by flow cytometry; and f) comparing the signal obtained for eachfluorescent label. wherein a difference in the signals measured at step(e) indicates that the biological sample comprises said shed ectodomainof CD31.